Implant exosome and application thereof in inflammation resistance, oxidation resistance and bacterium resistance

Abstract

The invention discloses an implant-derived exosome which is prepared by the following steps: adding cleaned and crushed colored rice flour into a PBS (Phosphate Buffer Solution) with the pH value of 6-7.4, homogenizing at a high speed, adding alpha-amylase into rice milk, performing enzymolysis for 1-2 hours under the conditions that the pH value is 6-7.4 and the temperature is 90-97 DEG C, filtering, and standing filtrate overnight; carrying out gradient differential centrifugation at 4 DEG C, collecting supernate, filtering, carrying out ultracentrifugation on filtrate at 4 DEG C and 12000-15000g for 1.5-3 hours, and collecting precipitate; and re-suspending the precipitate, enriching by using an MWCO ultrafiltration tube with the molecular weight cut-off of 100kDa, collecting the precipitate at the bottom of the ultrafiltration tube, and re-suspending in a PBS buffer solution, thereby obtaining the product. The exosome can effectively inhibit the generation of inflammatory factors, has a good inhibition effect on various pathogenic bacteria, also has an anti-oxidation effect, and has application prospects in the fields of inflammation resistance, bacteria resistance, oxidation resistance and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of a plant-derived exosome with anti-inflammatory, antioxidant and antibacterial functions. BACKGROUND

[0002] The field of biological medicine is continuously deepening the research on new treatment methods. Inflammation, oxidative stress and bacterial infection are key factors for many diseases, and it is of great significance to develop bioactive substances with anti-inflammatory, antioxidant and antibacterial functions. Exosomes are nanoscale vesicles secreted by cells, which can carry various bioactive molecules and participate in intercellular signal transmission and physiological regulation. Plant-derived exosomes, i.e. exosomes extracted from plants, have the advantages of wide source, high biological safety and low immunogenicity, and research shows that they have biological activities such as anti-inflammatory, antioxidant and antibacterial, providing a new idea for the research and development of new therapeutic drugs.

[0003] Inflammation is the immune response of the body to injury or pathogen invasion, and moderate inflammation helps to eliminate pathogens and repair tissues, but excessive inflammation can cause tissue damage and disease aggravation. Bacterial infection is a global health problem, and the increase in antibiotic resistance makes treatment more difficult, so it is urgent to develop new antibacterial agents.

[0004] At present, the research on plant-derived exosomes is in the early stage, the extraction method is complex, the yield is low, and the function verification is mostly carried out in vitro, and the in vivo anti-inflammatory, antioxidant and antibacterial mechanism research is not deep, which limits the clinical application transformation. SUMMARY

[0005] To solve the above technical problems, the application provides a plant-derived exosome with anti-inflammatory, antioxidant and antibacterial functions, which is prepared by the following steps: 1) Pretreatment of plant samples: first rinse the colored rice with deionized water and drain, then finely pulverize it; 2) Homogenization: add PBS buffer with pH 6-7.4 to the finely pulverized colored rice powder, and homogenize at high speed for 8-10 times (1-2 min / time); The high-speed homogenization is treated by a vortex oscillator at a speed of 1000-2000 rpm / min for 1-2 min; 3) Enzymatic treatment: add α - amylase to the rice solution in step 2) at a ratio, and enzymatically treat at pH 6-7.4 and 90-97℃ for 1-2 h, filter, and let the filtrate stand overnight; The mass-volume ratio g:mL of the colored rice powder to PBS buffer is 1:2-3, and the addition amount of alpha-amylase is 100-300 μg / 100 mL; 4) Differential centrifugation: after the enzyme solution is rested, the rice liquid is subjected to gradient differential centrifugation at 4 DEG C, the supernatant is collected, filtered, and the filtrate is subjected to ultracentrifugation at 4 DEG C, 12000-15000g for 1.5-3h, and the precipitate is collected; The gradient differential centrifugation is sequentially subjected to 900-1000g centrifugation for 8-10min, 1500-2000g centrifugation for 15-25min, 3500-4000g centrifugation for 35-45min, and 9500-10000g centrifugation for 55-65min; the filtration is performed using a 0.22pm filter; 5) Ultrafiltration concentration: the above precipitate is resuspended, enriched using a MWCO ultrafiltration tube with a molecular weight cut-off of 100kDa, the precipitate at the bottom of the ultrafiltration tube is collected, and resuspended in PBS buffer to obtain the phytogenic exosomes; The MWCO ultrafiltration tube is centrifuged at 4 DEG C, 3500g-5500g for 10-20min.

[0006] The phytogenic exosomes prepared by the present application have a diameter of 150-300nm and a zeta potential of -30--36mV.

[0007] The present application provides phytogenic exosomes with anti-inflammatory, antioxidant and antibacterial functions, which can comprehensively cope with various pathological states and provide a multi-effect solution for treating related diseases. The present application uses physical crushing, enzymatic hydrolysis, differential centrifugation, ultrafiltration and other means to significantly improve the yield and purity of exosomes, reduce production costs, and facilitate large-scale production and application. In addition, the present application broadens the plant species for exosome preparation, especially using colored rice with unique characteristics, providing more choices for the source of exosomes and enhancing the accessibility of resources. At the same time, the phytogenic exosomes have the advantages of wide source, high biological safety and low immunogenicity, reducing potential adverse reactions and improving the safety of clinical application.

[0008] The exosomes of the present application have broad application prospects in the fields of anti-inflammatory, antioxidant and antibacterial, providing a new direction for the development of biological and medical technology, and are expected to be used for treating various inflammatory diseases, oxidative stress related diseases and bacterial infections. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 The figure is a transmission electron micrograph of the exosomes of Example 1; the first line is a graph under a 100nm scale, the second line is a graph under a 200nm scale, and the third line is a graph under a 500nm scale; Figure 2 The figure is a nanoparticle tracking analysis (NTA) result graph of the exosomes of Example 1; Figure 3 The figure is a nanoparticle size (Zeta) potential graph of the exosomes of Example 1; Figure 4 This is a standard curve for bovine serum albumin concentration. Figure 5 The results show the scavenging rates of ABTS and DPPH free radicals by purple rice exosomes; Figure 6 The results of CCK-8 assay for the effect of purple rice exosomes on cell viability; Figure 7 The results of detecting the production of nitric oxide in cells by purple rice exosomes; Figure 8 Figure 1 shows the experimental results of detecting the levels of IL-6, IL-1β and TNF-α inflammatory factors in cell culture supernatant by ELISA. Figure 9 The figure shows the results of the exosome antibacterial assay using the inhibition zone method. Detailed Implementation

[0010] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Example 1: Preparation of plant-derived exosomes 1. Rinse the purple rice with deionized water and drain it. Then, use an air jet mill to pulverize it into a fine powder to 110-180mm. 2. Add PBS with pH 6.0 to 100g of ultrafine purple rice powder and homogenize it 8 times (1min / time) at 1500rpm / min using a vortex mixer. 3. Prepare the solution at a ratio of 200 μg / 100 mL. α- Amylase was added to the solution from step 2 and hydrolyzed at pH 6.0 and 97°C for 2 hours. The solution was then filtered through eight layers of gauze and allowed to stand overnight. 4. After the rice liquid has been hydrolyzed and allowed to stand, it is centrifuged at a low temperature and a high speed using a differential centrifuge. Specifically, at 4°C, the centrifugation is performed at 1000g for 10 min, 2000g for 20 min, 4000g for 40 min, and 10000g for 60 min. The supernatant is collected, filtered through a 0.22 μm filter, and the filtrate is then centrifuged at 12000g for 2 h at 4°C. The precipitate is collected. 5. Resuspend the precipitate from step 4 and enrich it using an MWCO ultrafiltration tube with a molecular weight cutoff of 100 kDa. Collect the precipitate at the bottom of the ultrafiltration tube and resuspend it in 1 mL of PBS buffer to obtain the purple rice exosome suspension (OEVs). 6. Transmission electron microscopy (TEM, JEOL, JEM-1400) was used to detect the morphology of exosomes. First, 8 μL of the exosome suspension from step 5 was dropped onto a copper grid, and after 3 min of standing, the excess liquid was carefully absorbed with filter paper. After repeating the sample dropping 3 times, 8 μL of 1% (w / v) uranyl acetate solution was added for negative staining. After 3 min of standing, the excess liquid was absorbed with filter paper, and then washed twice with double distilled water. After natural drying, the sample morphology was observed under a transmission electron microscope and photographed.

[0011] The results of transmission electron microscopy observation are shown in Figure 1 . In the experiment, the sample was photographed under the scales of 500, 200 and 100 nm, respectively. The results showed that the exosomes had obvious tea-tray-like structure, and the interface was clean, and no impurities were observed. This result showed that the extracted exosomes had typical vesicle characteristics in morphology, and the purity was high, and was not obviously contaminated.

[0012] 7. In the nanoparticle tracking analysis (NTA) detection, first, the sample pool was cleaned with deionized water, and then the instrument was calibrated with 100 nm polystyrene microspheres. Then the sample pool was cleaned again with 1x PBS buffer. The sample was diluted with 1x PBS buffer and then detected. The results are shown in Figure 2 . According to the results reported by NTA, there were two main particle size peaks in the sample, located at 150.1 nm and 263.6 nm, respectively, indicating that the sample had a bimodal particle size distribution. The volume median of the sample was 154.1 nm, and the volume span was 65.2. The concentration of the sample on the machine was 5.2x10 7 particles / mL (after dilution), and the original concentration was 2.8x10 10 particles / mL. These data showed that the particle size distribution of the sample was relatively concentrated, and the main particle size range met the typical size requirements of exosomes, suggesting that the sample may contain rich exosome components.

[0013] 8. Zeta potential detection, first, the sample pool was cleaned with purified water, and the instrument was calibrated with 100 nm polystyrene microspheres. The sample was diluted with purified water and then detected. The results of Zeta potential detection are shown in Figure 3 . The results showed that the Zeta potential of the sample was (-35.25±0.48) mV, indicating that the particles in the sample were negatively charged. The concentration of the sample on the machine was 3.5x10 7 particles / mL (after dilution), and the original concentration was 2.8x10 10particles / mL. These results provide important reference data for the charge characteristics and concentration analysis of exosomes. Zeta potential is an important indicator for measuring the stability of particles in solution. The higher the absolute value of the Zeta potential value, the more stable the system; on the contrary, the lower the absolute value, the system tends to condense or aggregate. In general, an absolute value of the Zeta potential value greater than 30 mV indicates that the sample system is stable, and less than 30 mV indicates that the sample system is unstable and prone to aggregation.

[0014] In this experiment, the absolute value of the Zeta potential of the sample was 35.25 mV, which was greater than 30 mV, indicating that the sample system had high stability and was not prone to aggregation or precipitation.

[0015] 9. BCA method for detecting the concentration of exosomes Take 1 μL, 2 μL, 4 μL of exosome suspension from step 5 and 19 μL, 18 μL, 16 μL of PBS (pH = 6.0) respectively, add to the 96-well plate, set 3 repeated holes to ensure the reliability of the data. Then add 200 μL of BCA working solution to each well and mix well. At the same time, prepare a series of known concentration bovine serum albumin, BSA standard protein solution with a gradient of 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 μg / mL, follow the same steps for processing as a control for the standard curve. Place the 96-well plate in a preheated 37°C incubator for 30 min to allow the reaction to proceed fully. After incubation, use a microplate reader to measure the absorbance of each well at a wavelength of 562 nm; The results are shown in Figure 4 , which shows the standard curve generated by the BCA method, with a linear equation of y = 0.3538 x + 0.1093, and a correlation coefficient R 2 = 0.9972, R > 0.99, indicating that the experimental results are reliable and can be used to accurately determine the concentration of unknown samples; The optical density (OD) values and corresponding concentrations (mg / mL) of the exosome suspension under different dilution ratios are listed in Table 1. The results under different dilution ratios are stable, further verifying the reliability of the experimental results; Table 1 BCA method for calibrating exosome concentration .

[0016] Example 2: Detection of the antioxidant capacity of purple rice exosome suspension (OEVs) in Example 1 1. ABTS method for antioxidant analysis Take (20- xμL PBS (pH=6.0) was added to the 96-well plate, and the sample solution was added x μL ( x The amount of sample solution was determined according to the preliminary test results, i.e. 0.5, 1, 2, 4 μL, then 80 μL of ABTS solution was added, mixed, reacted at 37°C in the dark for 6 min, and the absorbance was measured at 734 nm. The ABTS scavenging rate was calculated according to the following formula: ABTS· scavenging rate = ( A 0- A ) / A 0 x 100% In the formula: A 0 is the absorbance of 20 μL PBS (pH=6.0) and 80 μL ABTS; A is the absorbance of 20 μL of the sample with different dilution ratios and 80 μL ABTS.

[0017] 2. DPPH method for antioxidant analysis (20- x ) μL PBS (pH=6.0) was added to the 96-well plate, and the sample solution was added x μL ( x The amount of sample solution was determined according to the preliminary test results, i.e. 0.5, 1, 2, 4 μL, then 80 μL of ABTS solution was added, mixed, reacted at 37°C in the dark for 6 min, and the absorbance was measured at 734 nm. The ABTS scavenging rate was calculated according to the following formula: DPPH scavenging rate = ( A 0- A ) / A 0 x 100% In the formula: A 0 is the absorbance of 20 μL PBS (pH=6.0) and 80 μL DPPH; A is the absorbance of 20 μL of the sample with different dilution ratios and 80 μL DPPH; The results are shown in Figure 5As shown in the figure, the scavenging capacity of exosomes against DPPH free radicals was 73.40% after a 5-fold dilution, 67.27% after a 10-fold dilution, 40.31% after a 20-fold dilution, and 20.78% after a 40-fold dilution. In the ABTS free radical scavenging experiment, the scavenging capacity of exosomes against ABTS free radicals was 49.64% after a 5-fold dilution, 35.01% after a 10-fold dilution, 21.23% after a 20-fold dilution, and 13.34% after a 40-fold dilution. These results indicate that OEVs possess significant antioxidant activity, and their free radical scavenging capacity gradually decreases with increasing dilution factor. Although the scavenging rate decreases with increasing dilution factor, OEVs still maintain a certain antioxidant effect even at higher dilution factors. This suggests that OEVs have potential application value in antioxidant applications.

[0018] Example 3: Detection of the anti-inflammatory ability of purple rice exosome suspension (OEVs) from Example 1 1. Cell viability was determined using the CCK8 assay. RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin antibiotics; the cells were then incubated at a constant temperature of 37°C in a 5% CO2 incubator. When the cell culture reaches 80%–90% confluence, passage the cells, discard the culture medium, wash the cells with PBS to remove residual culture medium and serum, add an appropriate amount of PBS (pH=7.4) to the wash dish, gently pipette to remove all cells, and continue pipetting until a single-cell suspension is formed. Count the cells using a cell counter and adjust the cell density to 1×10⁻⁶. 5 Cells were seeded at 100 μL / well in 96-well plates (to eliminate edge effects, 100 μL of PBS (pH=7.4) was added to the outermost ring of the 96-well plate). After gentle mixing, the plates were incubated at 37°C and 5% CO2 for 24 h. The culture medium was then removed, and the cells were treated with different concentrations of OEVs. After 6 h, 100 μL of medium containing 10% CCK8 solution was added to each well, and the plates were incubated at 37°C and 5% CO2 for another 1 h. A blank group (no cells were seeded, only DMEM complete medium) and a control group (cells were seeded without exosomes) were also set up. The absorbance was measured at 450 nm, and the cell viability was calculated based on the absorbance using the following formula: Cell viability (%) = (OD1 - OD0) / (OD2 - OD0) In the formula: OD0 represents the absorbance value of the blank group, OD1 represents the absorbance value of the sample group, and OD2 represents the absorbance value of the control group; Cell viability results as follows Figure 6As shown in the figure, with the decrease of the concentration of the compound, the cell survival rate was significantly improved, and at the concentration of 1500 μg / mL, the cell survival rate was only 20.31%, showing strong cytotoxicity; when the concentration was reduced to 750 μg / mL, the cell survival rate increased to 29.88%, but the cell damage was still significant. Further reducing the concentration to 500 μg / mL, the cell survival rate was significantly improved to 88.75%, indicating that the toxicity of the compound at this concentration to the cells was greatly reduced. At the concentrations of 300 and 100 μg / mL, the cell survival rates were 97.47% and 106.51%, respectively, close to or even slightly higher than the untreated control group, suggesting that the compounds at these concentrations may have no toxicity to the cells, and even may have a slight promotion effect on cell proliferation.

[0019] 2. Determination of nitric oxide (NO) production RAW264.7 was cultured according to the above method and inoculated at 1×10 5 After the cells were cultured for 24 h and completely adhered, the medium was removed, and OEVs were added with a prepared solution at a concentration of 250, 125, and 62.5 μg / mL, and a positive drug dexamethasone (DXM) 1 μg / mL. The blank wells and model wells were replaced with culture medium, and the cells were pretreated for 6 h. Then, the cell supernatant was removed and the cells were carefully washed with PBS. Subsequently, except that the blank wells were replaced with culture medium, 2 mL of 1 μg / mL LPS was added to each of the other wells, and the cells were further cultured in the incubator for 18 h. After the culture was completed, the culture solution was collected, and the cells and debris were removed by centrifugation at 1000 rpm to obtain the supernatant. The inhibition of OEVs on the production of NO by LPS-induced RAW264.7 cells was determined according to the method described in the NO determination kit. 3. Determination of the expression level of inflammatory factors by ELISA RAW264.7 cells were cultured and treated with OEVs according to the method in step 2. After the cell culture was completed, the supernatant was collected, centrifuged at 5500 rpm to remove cell debris, and then measured. The effect of OEVs on the production of inflammatory factors by LPS-induced RAW264.7 cells was determined according to the method described in the ELISA determination kit. The specific determination method is as follows: (1) Double antibody sandwich: Take out the ELISA kit stored at 4°C and balance to room temperature. (The used well plate has been coated with a primary antibody and blocked.) Set up blank wells (without sample and enzyme-labeled reagent, and the rest of the steps are the same), standard wells, and sample wells. Add different concentrations of standard samples to each well containing a primary antibody at 50 μL / well. For sample wells, first add sample diluent (provided in the kit) 40 μL, and then add the sample to be tested 10 μL (the sample dilution is 5 times). Shake slowly and evenly, cover all the reaction wells with a sealing film, and incubate at 37°C for 30 min. (2) Washing the plate: After incubation, gently shake out the reaction solution in the wells and pat dry on a thick stack of absorbent paper. Add 350 μL of the prepared washing solution to each well, let stand for 30 seconds, then gently shake out the liquid and blot dry with absorbent paper. Repeat 5 times. (3) Enzyme conjugation: Add 50 μL of enzyme conjugation working solution to each well, and gently shake left and right to mix evenly. Then seal all the reaction wells and incubate at 37°C in the dark for 30 min; (4) Washing the plate: After incubation, wash the plate 5 times according to step (3); (5) Color development: Add 50 μL of color developer A to each well, then add 50 μL of color developer B, incubate at 37°C in the dark for 10 min, and observe the color change. (6) Termination of reaction: Add 50 μL of reaction termination solution to each well, mix well and observe the color reaction. When the color of the reaction solution in the well changes from blue to yellow, the reaction is terminated. Then measure the absorbance at a wavelength of 450 nm. The content of inflammatory factors in cells of each group of samples is calculated based on the standard curve plotted with the standard. The results are as follows Figure 7 , 8 All experimental data are expressed as "mean ± standard deviation (Mean ± SD)" (sample size ≥ 3), and one-way ANOVA was used. When the significance level ( p A value <0.05 was considered statistically significant. All analyses were performed using GraphPad Prism 10.1.2.

[0020] Compared with the untreated control group, the LPS-induced RAW264.7 cell model group showed higher levels of NO, IL-6, and TNF-α. α and IL-1 β The amount of production increased significantly ( P The concentration of LPS-induced anti-inflammatory cytokines (<0.0001) confirmed the successful establishment of an in vitro anti-inflammatory model of RAW264.7 cells. Furthermore, when different concentrations of purple rice exosomes were added to the LPS-induced model group, the production of NO, IL-6, TNF-α, and IL-1β showed a dose-dependent decreasing trend compared to the model group. This finding suggests that purple rice exosomes may exert their anti-inflammatory effects by inhibiting the production of inflammatory factors.

[0021] Example 4: Antibacterial effect test of purple rice exosome suspension (OEVs) from Example 1 The antibacterial activity of the exosomes prepared in Example 1 against Escherichia coli and Staphylococcus aureus was evaluated using inhibition zones. (1) Filter paper pre-treatment: Put the drug sensitive filter paper into 3 mg / mL outer upper (purple rice exosome suspension diluted 2 times) and outer lower (purple rice exosome suspension), 10 μg / mL ciprofloxacin (positive drug) respectively for 2 h and dry, each group do 3 parallel repeats; At the same time, set up a negative control group (without adding exosomes); (2) Dilution coating: evenly coat the diluted 1000 times of Staphylococcus aureus liquid and Escherichia coli liquid on the agar solid medium prepared in advance.

[0022] (3) Drug placement: Then put the above soaked and dried drug sensitive paper into the designated area of the culture medium.

[0023] (4) Culture: Finally, put it into a 37℃ constant temperature incubator for 24 h, observe the growth of the inhibition zone and measure the size of the inhibition zone, and analyze the bacteriostatic effect.

[0024] The experimental results are as follows Figure 9 , the exosomes have obvious bacteriostatic effect on Escherichia coli and Staphylococcus aureus. The control group (negative) Staphylococcus aureus and Escherichia coli colonies cover the agar plate; On the contrary, the area with exosome filter paper has obvious bacteriostatic ring. Among them, OEVs and diluted 2 times OEVs both form obvious bacteriostatic ring, and the size is similar but smaller than the positive control group, still showing that the exosomes have significant bacteriostatic property.

[0025] The preferred embodiments of the application are described in detail above, but the application is not limited thereto. Within the technical concept of the application, various simple modifications can be made to the technical solutions of the application, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be considered as disclosed by the application and fall within the protection scope of the application.

Claims

1. A plant-derived exosome derived from colored rice, characterized by: The washed and crushed colored rice powder was added to PBS buffer with pH 6~7.4 and homogenized at high speed, and then α - amylase was added to the rice slurry, and enzymolysis was carried out at pH 6~7.4 and 90~97℃ for 1~2h, filtration was performed, and the filtrate was left overnight; then gradient differential centrifugation was carried out at 4℃, the supernatant was collected, filtration was performed, the filtrate was ultracentrifuged at 4℃ and 12000~15000g for 1.5~3h, and the precipitate was collected; the precipitate was resuspended and enriched by using a MWCO ultrafiltration tube with a molecular weight cutoff of 100kDa, the precipitate at the bottom of the ultrafiltration tube was collected, and resuspension in PBS buffer obtained the plant-derived exosomes.

2. The phytoncide exosome of claim 1, wherein: High-speed homogenization is using vortex shaker at 1000-2000 rpm / min for 1-2 min, repeated 8-10 times.

3. The phytoncide exosome of claim 1, wherein: The mass-volume ratio of colored rice powder to PBS buffer is 1:2-3, and the amount of α-amylase added is 100-300 μg / 100 mL.

4. The phytoncide exosome of claim 1, wherein: Gradient differential centrifugation is sequentially subjected to 900-1000 g centrifugation for 8-10 min, 1500-2000 g centrifugation for 15-25 min, 3500-4000 g centrifugation for 35-45 min, and 9500-10000 g centrifugation for 55-65 min; filtration is performed using a 0.22 μm filter.

5. The phytoncide exosome of claim 1, wherein: The MWCO ultrafiltration tube is centrifuged at 4℃, 3500g-5500g for 10-20 min.

6. The phytoncide exosome of claim 1, wherein: Phytogenic exosomes have a diameter of 150-300 nm and a ζ potential of -30 to -36 mV.

7. Use of the phytogenic exosomes of any one of claims 1-6 in the preparation of anti-inflammatory drugs.

8. Use of the phytogenic exosomes of any one of claims 1-6 in the preparation of antioxidant drugs.

9. Use of the phytonic exosome of any one of claims 1-6 in the preparation of a formulation against Escherichia coli (E. coli) Escherichia coli ) and Staphylococcus aureus (S. aureus) Staphylococcus aureus ).

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