Application of iturin in relieving drug resistance of silver nanoparticles
By making iturin-AgNPs, the problem of silver nanoparticles induced bacterial resistance was solved, and the resistance to E. coli was effectively eliminated, providing a new idea to prevent bacterial resistance.
Patent Information
- Application Number
- CN202411672071.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-21
- Publication Date
- 2025-02-21
AI Technical Summary
Silver nanoparticles induce bacterial resistance when inhibiting bacteria, and lack the technology and products to relieve this resistance.
The Bacillus subtilis metabolite is made into Iturin-AgNPs, and through specific preparation methods and treatment steps, composite nanoparticles with a particle size of 21±7nm and a silver element content of 145μg/mg were formed.
Iturin-AgNPs can effectively relieve E. coli from drug resistance to commercial silver nanoparticles (PVP-AgNPs), providing a new idea to prevent bacteria from drug resistance to silver nanoparticles.
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Abstract
Description
Technical Field
[0001] The invention relates to a new application efficacy field of iturin, a metabolite of Bacillus subtilis, and in particular to an application of iturin, a metabolite of Bacillus subtilis, in removing drug resistance of silver nanoparticles. Background Art
[0002] Infections caused by pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella, and Candida albicans are common in clinical cases. The problem of drug-resistant bacteria caused by antibiotic treatment over the years has become an important factor threatening human health and life. Finding and developing new, safe, and effective drugs to inhibit drug-resistant bacteria is an important challenge facing the international medical community.
[0003] AgNPs (silver nanoparticles) are one of the antibacterial drugs with the broadest antibacterial spectrum reported so far. It can not only effectively inhibit a variety of bacteria, but also has a certain inhibitory effect on a variety of fungi, showing good application prospects. However, reports in recent years have pointed out that silver nanoparticles can induce bacterial resistance. Although the corresponding phenomena and mechanisms are widely discussed in the literature, there is a lack of research on the technology and products to eliminate this resistance. Summary of the invention
[0004] The present invention aims to provide a new application of iturin for eliminating the drug resistance of silver nanoparticles.
[0005] The technical solution adopted by the present invention is:
[0006] The application of iturin in relieving the resistance of silver nanoparticles, and iturin was made into Iturin-AgNPs.
[0007] Furthermore, the Iturin-AgNPs have a particle size of 21±7 nm and a silver content of 145 μg / mg.
[0008] Furthermore, the preparation method of Iturin-AgNPs comprises the following steps:
[0009] (1) Weighing the Iturin powder obtained by freeze-drying and dissolving it in deionized water to obtain an Iturin solution with a concentration of 1 mg / mL;
[0010] (2) adjusting the pH of the Iturin solution to 9-10 with sodium hydroxide solution (1 M) and nitric acid solution (1 M), and stirring to make the solution fully mixed;
[0011] (3) weighing silver nitrate powder and dissolving it in deionized water to obtain a silver nitrate solution with a concentration of 2 mg / mL;
[0012] (4) Slowly add the silver nitrate solution to the Iturin solution at a volume ratio of 1:10, stirring continuously to prevent the formation of silver hydroxide precipitation;
[0013] (5) The mixed solution was irradiated under a 365 nm ultraviolet lamp for 2 h, with the light source 5 cm away from the liquid surface. The reaction mixture gradually changed from colorless to brownish yellow, indicating that silver nanoparticles were synthesized.
[0014] (6) The reaction product was centrifuged at 10,000 rpm and 4°C for 30 min and the supernatant was discarded;
[0015] (7) Wash the precipitate twice with deionized water, and centrifuge under the same conditions as step (6);
[0016] (8) Resuspend in an appropriate amount of deionized water to obtain Iturin-AgNPs solution, which is stored in a refrigerator at 4 °C;
[0017] (9) Freeze-drying to obtain Iturin-AgNPs.
[0018] The beneficial technical effects of the present invention are as follows:
[0019] (1) The present invention discloses the application of iturin in eliminating the drug resistance of silver nanoparticles. Iturin-AgNPs are prepared from iturin, and compared with chemically synthesized PVP-AgNPs, iturin-AgNPs are less likely to cause drug resistance in Escherichia coli.
[0020] (2) The iturin-AgNPs prepared in the present invention can eliminate the resistance of Escherichia coli to commercial silver nanoparticles (PVP-AgNPs), providing a new idea for preventing bacteria from developing resistance to silver nanoparticles. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 .(A) Characterization of PVP-AgNPs and iturin-AgNPs, and the intracellular protein and nucleic acid content of Escherichia coli incubated with iturin-AgNPs (1 / 2MIC, MIC, 2MIC, 4MIC); (B) ROS and PI staining of Escherichia coli treated with iturin-AgNPs (MIC) for different times (0h, 1h, 2h, 4h); (C) Scanning electron microscopy and transmission electron microscopy of Escherichia coli cells treated with iturin-AgNPs.
[0022] Figure 2.(A) MIC and intracellular element analysis of PVP-AgNPs and IturinA-AgNPs against drug-resistant Escherichia coli; (B) TEM and EDS analysis of Escherichia coli after treatment with IturinA-AgNPs or PVP-AgNPs; (C) zeta potential (mV) of Escherichia coli after treatment with IturinA-AgNPs or PVP-AgNPs; (D) Fluorescence intensity (au) of Escherichia coli after treatment with IturinA-AgNPs or PVP-AgNPs.
[0023] Figure 3 (A) Relative position of the fli gene on the E. coli chromosome; (B) Flagellin expression level and genes involved in flagellin biosynthesis (gene names marked in green and red represent downregulated and upregulated genes, respectively); (C) Expression of genes related to flagellin synthesis after different treatment times (S: sensitive E. coli; R: resistant E. coli).
[0024] Figure 4 Effects of PVP-AgNPs and IturinA-AgNPs on the efflux vesicles of sensitive and resistant Escherichia coli: (A) Relative position of the mlaA-F gene on the chromosome; (B) PVP-AgNPs treatment of resistant Escherichia coli efflux vesicles; (C) EDS-TEM analysis of sensitive and resistant Escherichia coli treated with PVP-AgNPs and IturinA-AgNPs; (D) Effects of PVP-AgNPs and IturinA-AgNPs on the expression of Mla pathway genes in sensitive and resistant Escherichia coli; (E) MICs of PVP-AgNPs and IturinA-AgNPs for ΔmlaA and mlaA+ Escherichia coli; (F) Fluorescence intensity (au) of ΔmlaA and mlaA+ Escherichia coli treated with IturinA-AgNPs or PVP-AgNPs. ΔmlaA gene knockout bacteria; +mlaA gene overexpression bacteria. * indicates p<0.05, the difference between the treated group and the blank control group is significant, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001 (n=3).
[0025] Figure 5 .Effects of PVP-AgNPs and Iturin A-AgNPs on Escherichia coli proteins: (A) Protein polyacrylamide gel of sensitive and resistant bacteria bound to PVP-AgNPs and Iturin A-AgNPs; (B) Effects of different treatments on the grayscale of the bands; (C) Mass spectrometry identification of IturinA-AgNPs bound to resistant bacteria proteins.
[0026] Figure 6Effects of PVP-AgNPs and Iturin A-AgNPs on energy metabolism of Escherichia coli: (A) Main down-regulated pathways in KEGG in RC vs SC and main up-regulated pathways in KEGG in RI vs RC; RC: PVP-AgNPs treatment of silver nanoparticle-resistant bacteria; SC: PVP-AgNPs treatment of silver nanoparticle-sensitive bacteria; RI: iturin-AgNPs treatment of silver nanoparticle-resistant bacteria; (B) GO analysis of genes in RC vs SC (A) and RI vs RC (B); (C) Relative positions of energy metabolism-related genes atpC, -G, and -H on chromosomes and the effects of PVP-AgNPs and Iturin A-AgNPs on the expression of energy metabolism genes in Escherichia coli. DETAILED DESCRIPTION
[0027] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0028] The invention discloses an application of iturin in eliminating the drug resistance of silver nanoparticles. Specifically, iturin is prepared into Iturin-AgNPs. The particle size of the Iturin-AgNPs is 21±7nm and the silver content is 145μg / mg.
[0029] Example 1
[0030] The preparation method of Iturin-AgNPs comprises the following steps:
[0031] (1) Weighing the Iturin powder obtained by freeze-drying and dissolving it in deionized water to obtain an Iturin solution with a concentration of 1 mg / mL;
[0032] (2) adjusting the pH of the Iturin solution to 9-10 with sodium hydroxide solution (1 M) and nitric acid solution (1 M), and stirring to make the solution fully mixed;
[0033] (3) weighing silver nitrate powder and dissolving it in deionized water to obtain a silver nitrate solution with a concentration of 2 mg / mL;
[0034] (4) Slowly add the silver nitrate solution to the Iturin solution at a volume ratio of 1:10, stirring continuously to prevent the formation of silver hydroxide precipitation;
[0035] (5) The mixed solution was irradiated under a 365 nm ultraviolet lamp for 2 h, with the light source 5 cm away from the liquid surface. The reaction mixture gradually changed from colorless to brownish yellow, indicating that silver nanoparticles were synthesized.
[0036] (6) The reaction product was centrifuged at 10,000 rpm and 4°C for 30 min and the supernatant was discarded;
[0037] (7) Wash the precipitate twice with deionized water, and centrifuge under the same conditions as step (6);
[0038] (8) Resuspend in an appropriate amount of deionized water to obtain Iturin-AgNPs solution, which is stored in a refrigerator at 4 °C;
[0039] (9) Take 1 mL of the prepared Iturin-AgNPs solution and freeze-dry it to obtain Iturin-AgNPs. Then weigh it and calculate the solution concentration and yield.
[0040] Embodiment 2:
[0041] The Iturin-AgNPs preparation method comprises the following steps:
[0042] (1) The Iturin powder obtained by freeze-drying was weighed and dissolved in deionized water to obtain a lipopeptide solution with a concentration of 1 mg / mL.
[0043] (2) Adjust the pH of the lipopeptide solution to 9-10 with sodium hydroxide solution (1 M) and nitric acid solution (1 M), and stir to make the solution fully mixed.
[0044] (3) Weigh silver nitrate powder and dissolve it in deionized water to obtain a silver nitrate solution with a concentration of 2 mg / mL.
[0045] (4) Slowly add the silver nitrate solution to the lipopeptide solution (volume ratio of 1:10) and stir continuously to prevent the formation of silver hydroxide precipitation.
[0046] (5) The mixed solution was irradiated under an ultraviolet lamp (365 nm) for 2 h, with the light source being 5 cm away from the liquid surface. The reaction mixture gradually changed from colorless to brown-yellow, indicating that silver nanoparticles were synthesized.
[0047] (6) Centrifuge the reaction product (10,000 rpm, 30 min, 4°C) and discard the supernatant.
[0048] (7) Wash the precipitate twice with deionized water, and centrifuge under the same conditions as step (6);
[0049] (8) Resuspend in an appropriate amount of deionized water to obtain Iturin-AgNPs solution, which was stored in a 4°C refrigerator.
[0050] (9) Take 1 mL of the prepared Iturin-AgNPs solution, freeze-dry it, and weigh it to calculate the solution concentration and yield.
[0051] The characterization results of the obtained silver nanoparticles are as follows Figure 1As shown in the figure, the particle size of Iturin-AgNPs (21±7nm) is very similar to that of PVP-AgNPs (20±6nm), but the Ag content of Iturin-AgNPs (145μg / mg) is about 1 / 5 of that of PVP-AgNPs (715μg / mg). Nevertheless, the MIC of Iturin-AgNPs against E. coli is 2.5μg / mL, which is the same as that of PVP-AgNPs.
[0052] 1. The study plan of the inhibitory effect of Iturin-AgNPs on Escherichia coli is as follows:
[0053] Take the E. coli in the logarithmic growth phase, centrifuge at 6000rpm for 10min, collect the bacterial precipitate, and resuspend it with PBS solution (0.01M, pH 7.3). After washing three times, discard the residual medium. Take the bacterial precipitate and resuspend it with 2mL PBS (0.01M, pH 7.3). After counting, dilute it to 1×10 with PBS (0.01M, pH 7.3) solution. 9 / ml. Then add different concentrations of iturin-AgNPs solution (final concentration in culture medium is 0, 1 / 2MIC, MIC, 2MIC, 4MIC), all inoculates are placed at 200rpm and 37℃ for culture, take out the culture after 4h, centrifuge at 6000rpm for 10min, take the supernatant, determine the protein concentration in the supernatant by BCA method, and determine the DNA and RNA concentration in the supernatant by UV spectrophotometer. Use PI staining combined with fluorescence microscopy to observe the changes in cell membrane permeability. Use Reactive Oxygen Species Assay Kit to analyze whether iturin-AgNPs treatment will cause changes in ROS levels in bacteria. Transmission electron microscopy and scanning electron microscopy were used to observe changes in the interior and surface of the bacteria.
[0054] The inhibitory effect of Iturin-AgNPs on Escherichia coli Figure 1 As shown, iturin-AgNPs quickly entered the pathogen cells within 1 hour and then exited the cells in the form of vesicles, causing the leakage of intracellular proteins and nucleic acids, resulting in increased cell membrane permeability and increased ROS levels, ultimately leading to bacterial death.
[0055] 2. In order to explore whether the bacteria will develop tolerance to iturin-AgNPs and PVP-AgNPs, E. coli ATCC25922 was used as a model bacterium and was continuously exposed to sub-inhibitory concentrations of iturin-AgNPs and PVP-AgNPs. The specific steps are as follows:
[0056] Take Escherichia coli cultured in LB medium to the logarithmic growth phase, collect the cells by centrifugation, and adjust the cell density after appropriate dilution to make the corresponding concentration of 1×10 6 CFU / mL, and then transferred to a 96-well plate already filled with a certain amount of LB medium. 0.1mL of different concentrations (0.625, 1.25, 2.5, 5 and 10μg / mL) of iturin-AgNPs or PVP-AgNPs were added to each well of the plate. The uninoculated culture medium was used as the negative control, and the positive control was only inoculated with Escherichia coli without adding antibacterial agents. All cultures were cultured at 37°C for 24h, and the growth of bacteria in each well was observed. The lowest concentration that could completely inhibit the growth of Escherichia coli was the MIC value of the sample used. After each round of culture, 10μL of LB culture medium containing surviving bacteria was taken from each of the three wells with sub-inhibitory concentrations, mixed, and inoculated in 5mL LB culture medium. After continuing to subculture at 37°C for 12h, it was inoculated again into a 96-well plate containing different concentrations of drugs, and then the next round of culture was carried out. From the initial inoculation to the preparation of the new inoculum, the entire process is considered to have completed a cycle of bacterial resistance culture.
[0057] Those bacteria that caused the MIC to increase after completing a certain cycle were considered to have developed resistance. The results are shown in Table 1. After 22 cycles of culture, Escherichia coli had developed tolerance to PVP-AgNPs, but not to Iturin-AgNPs.
[0058] Table 1 MIC values of Escherichia coli during 22 cycles of continuous exposure to PVP-AgNPs and Iturin-AgNPs
[0059]
[0060] 3. Inhibitory effects of PVP-AgNPs and IturinA-AgNPs on sensitive and resistant Escherichia coli
[0061] The changes in the MIC value of Escherichia coli after AgNPs treatment were determined by well plate; the changes in the internal structure and element distribution of Escherichia coli after treatment with different AgNPs were analyzed by EDS-TEM; the temporal changes in the Zeta potential of Escherichia coli after AgNPs treatment were measured (0min, 10min, 20min, 30min, 60min, 90min), and the changes in the surface charge of Escherichia coli and the effects of AgNPs on the stability of the Escherichia coli system were analyzed; the accumulation of intracellular ROS in Escherichia coli after AgNPs treatment for 1h, 2h, and 4h was quantitatively characterized by a reactive oxygen species (ROS) detection kit (ROS Assay Kit), and whether the resistance of Escherichia coli to AgNPs was related to the inhibition of intracellular ROS production was analyzed.
[0062] The results are as follows Figure 2 As shown in the figure, iturin-AgNPs can relieve the drug resistance of Escherichia coli caused by PVP-AgNPs, and iturin-AgNPs can enter the cells within 1 hour; in the early stage of treatment, the amount of protein leakage is greater than the amount of nucleic acid leakage; the cells cannot completely expel the iturin-AgNPs that enter the cells. Drug-resistant bacteria slow down the rate of intracellular protein efflux caused by iturin-AgNPs to a certain extent. When PVP-AgNPs treat sensitive bacteria, it can cause rapid leakage of intracellular proteins within the first hour, but the leakage of nucleic acids is slower, resulting in a smaller increase in the relative abundance of silver nanoparticles entering the cells. When PVP-AgNPs are used to treat drug-resistant bacteria, the protein content in the cells does not decrease but increases, resulting in a continuous decrease in the relative abundance of nucleic acids and silver nanoparticles in the cells. The Zeta potential of drug-resistant bacteria treated with PVP-AgNPs first increased, which may be because drug-resistant bacteria prevent PVP-AgNPs from entering and exerting an antibacterial effect by expelling PVP-AgNPs; Iturin-AgNPs can make the absolute value of the Zeta potential of Escherichia coli decrease faster, that is, the time required for Iturin-AgNPs to take effect is shorter, which may be that Iturin-AgNPs inhibit the excretion of Iturin-AgNPs by Escherichia coli, so it can enter the cell faster to exert an antibacterial effect. In addition, the decrease in the absolute value of Zeta potential indicates that the permeability of the Escherichia coli membrane has increased, indicating that Iturin-AgNPs can disrupt the stability of the cell membrane faster, destroy the transmembrane potential, reduce cell viability, and cause cell death. PVP-AgNPs had no significant effect on the ROS production of Escherichia coli, while Iturin-AgNPs caused a very significant increase in the intracellular ROS of Escherichia coli, and the intracellular ROS of Escherichia coli increased continuously with time from 1h, 2h and 4h after the addition of Iturin-AgNPs. Therefore, the differences between PVP-AgNPs and Iturin-AgNPs in causing changes in intracellular ROS in Escherichia coli can explain the differences in their antibacterial mechanisms.
[0063] 4. Effects of iturin-AgNPs and PVP-AgNPs on the transcription and expression of bacterial flagellin
[0064] Sensitive bacteria E.coli ATCC25922 and resistant bacteria were cultured in 250mL LB medium for 16 hours. After reaching their logarithmic growth phase, the bacteria were collected by centrifugation, treated with iturin-AgNPs and PVP-AgNPs (at MIC concentration) for 4 hours, and centrifuged at 5000rpm for 20 minutes until all bacteria were precipitated. The precipitate was then collected, and a portion was subjected to transcriptome detection and analysis by Tianjin Novogene Bioinformatics Technology Co., Ltd. A portion was resuspended with 0.9% NaCl. The pH value was adjusted to 2, and after shaking in a magnetic stirrer for 30 minutes, the flagella were separated by centrifugation at 10000rpm for 40 minutes. The supernatant was then transferred to a new tube and the bacterial precipitate was discarded. The pH of the supernatant was adjusted to 7.0-7.2, and (NH4)2SO4 was added to the supernatant. After incubation at 4°C overnight, the supernatant was centrifuged at 12000 rpm for 40 min, and the precipitate was resuspended in 2 mL PBS to obtain flagellin. The concentration of the obtained flagellin was measured using a BCA kit.
[0065] The results are as follows Figure 3 As shown. It can be seen that after the drug-resistant bacteria were treated with PVP-AgNPs, the secretion of flagellin by the bacteria did increase significantly, while the treatment of sensitive bacteria did not lead to an increase in the secretion of flagellin. However, the treatment of drug-resistant bacteria with iturin-AgNPs did not cause an increase in the secretion of flagellin. The expression of flagellin synthesis genes in drug-resistant bacteria treated with PVP-AgNPs was significantly upregulated from 0 to 60 minutes, while the expression of flagellin synthesis genes in drug-resistant bacteria treated with Iturin-AgNPs was significantly downregulated from 0 to 30 minutes, and the inhibition of gene expression was not relieved at 240 minutes. Iturin-AgNPs can significantly inhibit the expression of flagellin synthesis genes in drug-resistant bacteria, thereby avoiding the aggregation of AgNPs caused by excessive expression of flagellin, while PVP-AgNPs will upregulate the expression of these genes in drug-resistant bacteria. This shows that the resistance of Escherichia coli to PVP-AgNPs is related to its induction of flagellin transcription and increased secretion.
[0066] 5. Effects of PVP-AgNPs and Iturin-AgNPs on the efflux vesicles of sensitive and resistant Escherichia coli
[0067] The Escherichia coli cell membrane was stained with FM4-64 fluorescent dye, and the vesicle extrusion phenomenon of the Escherichia coli cell membrane after AgNPs treatment was observed by laser scanning confocal microscopy. Real-time fluorescence quantitative PCR was used to determine the temporal changes in the expression of genes related to the Mla efflux vesicle pathway of sensitive and resistant Escherichia coli treated with PVP-AgNPs and Iturin A-AgNPs, respectively (0min, 10min, 20min, 30min, 60min, 120min, 240min), and the relationship between the expression of genes related to the Mla pathway and the generation and elimination of Escherichia coli resistance to AgNPs was analyzed.
[0068] The results are shown in Figure 4. PVP-AgNPs-resistant bacteria can expel AgNPs from cells through outer membrane vesicles and maintain the stability of the cell membrane. Overexpression of the mlaA gene improves the resistance of the bacteria to PVP-AgNPs, and knocking out the mlaA gene has no significant effect on the antibacterial effect of PVP-AgNPs. Iturin A-AgNPs can inhibit the formation of outer membrane vesicles of resistant bacteria and quickly inhibit the expression of Mla pathway genes. Knocking out the mlaA gene can increase the sensitivity of cells to Iturin A-AgNPs, while overexpression of the mlaA gene will increase the resistance of bacteria to Iturin A-AgNPs. This shows that outer membrane vesicle formation and the Mla pathway play an important role in causing Escherichia coli to resist PVP-AgNPs, and Iturin A-AgNPs can relieve the tolerance of bacteria by inhibiting the expression of genes related to the Mla pathway. At the same time, IturinA-AgNPs can significantly inhibit the expression of cell wall synthesis genes of tolerant bacteria and sensitive bacteria, but PVP-AgNPs has no inhibitory effect.
[0069] 6. Effects of PVP-AgNPs and Iturin-AgNPs on Escherichia coli proteins
[0070] A cell-free extract (CFX) rich in intracellular proteins of E. coli was prepared, and AgNPs were co-incubated with CFX to obtain intracellular proteins of E. coli bound to AgNPs. Proteins of different sizes were separated by SDS-PAGE electrophoresis, and proteins with high affinity to PVP-AgNPs and Iturin-AgNPs were identified by mass spectrometry (MS / MS). Figure 5 As shown, Iturin-AgNPs can bind to proteins related to DNA replication, protein synthesis, cell wall synthesis, material transport, and aerobic metabolism in resistant bacteria cells faster and more tightly than PVP-AgNPs, thereby causing disorders in material synthesis in cells and maintaining its antibacterial and bactericidal effects on resistant bacteria.
[0071] 7. Effects of PVP-AgNPs and Iturin-AgNPs on the Energy Metabolism of Escherichia coli
[0072] After PVP-AgNPs and Iturin-AgNPs were used to treat sensitive and resistant Escherichia coli, respectively, transcriptome detection and analysis were performed by Tianjin Novogene Bioinformatics Technology Co., Ltd., and real-time fluorescence quantitative PCR was used to determine the effects on the expression of energy metabolism-related genes. Figure 6 As shown in the figure, compared with sensitive bacteria, PVP-AgNPs-resistant bacteria can enter a "dormant" protection mode by downregulating the expression of genes related to energy metabolism, thereby resisting the damage of AgNPs materials to cells. In contrast, Iturin-AgNPs can upregulate the expression of genes related to energy metabolism in tolerant bacteria, breaking their "dormant" protection mode and leading to cell damage and death.
[0073] The content of the present invention is not limited to the embodiments listed, and any equivalent changes made to the technical solution of the present invention by ordinary technicians in this field after reading the description of the present invention are covered by the claims of the present invention.
Claims
1. The use of iturin in removing drug resistance of silver nanoparticles, characterized in that: Iturin was made into Iturin-AgNPs.
2. The use of iturin in removing drug resistance of silver nanoparticles according to claim 1, characterized in that: The Iturin-AgNPs have a particle size of 21±7 nm and a silver content of 145 μg / mg.
3. The use of iturin in removing drug resistance of silver nanoparticles according to claim 2, characterized in that: The preparation method of Iturin-AgNPs comprises the following steps: (1) Weighing the Iturin powder obtained by freeze-drying and dissolving it in deionized water to obtain an Iturin solution with a concentration of 1 mg / mL; (2) adjusting the pH of the Iturin solution to 9-10 with sodium hydroxide solution (1 M) and nitric acid solution (1 M), and stirring to make the solution fully mixed; (3) weighing silver nitrate powder and dissolving it in deionized water to obtain a silver nitrate solution with a concentration of 2 mg / mL; (4) Slowly add the silver nitrate solution to the Iturin solution at a volume ratio of 1:10, stirring continuously to prevent the formation of silver hydroxide precipitation; (5) The mixed solution was irradiated under a 365 nm ultraviolet lamp for 2 h, with the light source 5 cm away from the liquid surface. The reaction mixture gradually changed from colorless to brownish yellow, indicating that silver nanoparticles were synthesized. (6) The reaction product was centrifuged at 10,000 rpm and 4°C for 30 min and the supernatant was discarded; (7) Wash the precipitate twice with deionized water, and centrifuge under the same conditions as step (6); (8) Resuspend in an appropriate amount of deionized water to obtain Iturin-AgNPs solution, which is stored in a refrigerator at 4 °C; (9) Freeze-drying to obtain Iturin-AgNPs.