Surfactin-based self-assembled nano synergistic antibacterial system and application thereof

Abstract

The invention discloses a self-assembled nano synergistic antibacterial system constructed on the basis of Surfactin and application of the self-assembled nano synergistic antibacterial system in prevention and control of filamentous fungi. According to the preparation method, the excellent amphipathy of Surfactin is utilized, the emulsifying effect and the bactericidal synergistic effect of Surfactin are exerted at the same time, hydrophobic antibacterial active ingredients are self-assembled and wrapped to form stable nano-particles with the particle size being 20-200 nm and the PDI being smaller than or equal to 0.3 in the mass ratio of 1: 5 to 2: 1, and the FIC is smaller than or equal to 0.5 and the strong synergism is achieved. The system has the characteristic of dual purposes by one dose: Surfactin not only assists the hydrophobic antibacterial active component to penetrate through the fungal cell wall, but also cooperates with the hydrophobic antibacterial active component to trigger significant outbreak (5.8 times at most) of the active oxygen level in fungal cells, and cooperatively induces programmed death of the fungal cells. The hydrophobic active ingredients comprise plant essential oil (such as cinnamyl aldehyde), microbial source antibiotics (such as echinocandin) and broad-spectrum hydrophobic chemical bactericides (such as triazole and methoxy acrylate). The nano synergistic antibacterial system is simple in preparation process, full in biological source and free of chemical residues, the unexpected technical effects that the dosage of active ingredients is reduced by 70% or above, and the field control effect reaches up to 85% are achieved, the limitation of the known technology is remarkably overcome, and the nano synergistic antibacterial system has wide application prospects in the fields of green agriculture and food safety.

Description

Technical Field

[0001] This invention relates to the field of bio-based nano-drug delivery systems, specifically to a self-assembled nano-synergistic antibacterial system constructed using the specific synergistic mechanism of Surfactin, its preparation method, and its application in the prevention and control of filamentous fungi. Background Technology

[0002] Crop diseases caused by filamentous fungi (such as Fusarium head blight, gray mold, and root rot) and post-harvest spoilage of agricultural products (such as Aspergillus flavus and its aflatoxin contamination) have resulted in enormous economic losses. Currently, the main control methods still rely on chemically synthesized fungicides. Long-term use has led to serious drug resistance in pathogens, rendering existing fungicides gradually ineffective, while also causing environmental pollution and food safety issues.

[0003] Hydrophobic antibacterial active ingredients, including plant essential oils, microbial antibiotics, and some chemically synthesized fungicides, have the potential to replace chemical pesticides. However, these substances generally suffer from poor water solubility, low bioavailability, volatility, or low chemical stability, which severely limit their practical application.

[0004] Traditional methods generally rely on chemical surfactants such as Tween-80 or biosurfactants such as rhamnolipids as emulsifiers. However, existing technologies still have the following shortcomings: (1) wide particle size distribution and poor physical stability, which easily lead to flocculation or stratification; (2) low encapsulation rate of hydrophobic active ingredients and uneven release, resulting in unstable field control efficacy; (3) the relationship between the active ingredients and the emulsification system is mostly a simple mixing relationship, lacking clear evidence of synergistic effects, making it difficult to significantly reduce the amount of active ingredients used; (4) for essential oil active ingredients or some chemical fungicides, many emulsification systems show insufficient penetration of hyphae or spores, thus limiting the antibacterial effect; (5) existing emulsification systems are difficult to induce programmed cell death in filamentous fungi, and more often only show general growth inhibition, lacking breakthroughs at the in-depth mechanism level; (6) the use of chemical surfactants increases ineffective components and high chemical residues.

[0005] Surfactin is a cyclic lipopeptide produced by Bacillus, possessing excellent surface activity and some antibacterial ability. However, using Surfactin alone to control filamentous fungi requires large dosages and is costly. Furthermore, its independent control effect is limited by its inability to effectively penetrate fungal cell walls. Although some literature reports Surfactin possessing certain antibacterial and surface activity, it has not been observed as a dual-function component ("emulsifier + synergist") for constructing stable nanostructures, and there has been no research on its synergistic window with hydrophobic active ingredients or potential cell death signaling pathways.

[0006] In summary, existing technologies generally suffer from the following technical pain points:

[0007] (1) Hydrophobic antibacterial components are difficult to disperse stably, making it difficult to improve the bactericidal effect;

[0008] (2) Existing emulsion systems are unstable, such as unstable particle size and high PDI, making it difficult to exert stable drug efficacy;

[0009] (3) In the prior art, chemical surfactants such as Tween-80 or common biosurfactants such as rhamnolipids only play a physical emulsifying role and lack an active synergistic mechanism with hydrophobic active ingredients. Experiments show that the FIC index of systems constructed with common emulsifiers is usually >0.5 (indicating additive or unrelated effects), which cannot achieve true "synergistic effect and dosage reduction" and is difficult to induce deep-level programmed cell death in fungi.

[0010] (4) It lacks a deep bactericidal mechanism against filamentous fungi, such as inducing intracellular reactive oxygen species bursts and programmed cell death.

[0011] (5) There are no clear rules to be found regarding the proportion, particle size stability and synergistic window of the biosurfactant system;

[0012] (6) Single active ingredients have limited efficacy, require large dosages, and are costly;

[0013] (7) The existing technology as a whole lacks a comprehensive solution that can "simultaneously combine stability, synergy and mechanism enhancement". Summary of the Invention

[0014] This invention overcomes the shortcomings of existing emulsion systems, such as poor stability, lack of synergy, limited inhibitory effect on filamentous fungi, and difficulty in inducing programmed cell death in fungi. It provides a self-assembled nano-synergistic antibacterial system based on Surfactin, its preparation method, and its applications. By compounding Surfactin with hydrophobic antibacterial active ingredients at a specific mass ratio, a physically stable, controllable particle size, and significantly synergistic antibacterial nano-drug-loaded system is constructed, thereby achieving efficient, safe, and low-dose control of fungal diseases.

[0015] This invention provides a self-assembled nano-synergistic antibacterial system based on Surfactin, the system comprising:

[0016] Active ingredient A: Surfactin or its salt;

[0017] Active ingredient B: Hydrophobic antibacterial active ingredients, including plant essential oils, microbial antibiotics, or hydrophobic chemical bactericides.

[0018] The mass ratio of A to B is controlled to be 1:5-2:1.

[0019] Within this mass ratio range, Surfactin and hydrophobic antibacterial components can spontaneously form a uniform and stable oil-in-water (O / W) nanostructure through hydrophobic interactions, electrostatic interactions, and interfacial activation, and satisfy the following characteristics:

[0020] (1) The average particle size of the system measured by dynamic light scattering (DLS) is 20-200 nm;

[0021] (2) The polydispersity index (PDI) of the system is ≤0.30;

[0022] (3) The synergistic antibacterial index FIC determined by the checkerboard method is ≤0.5, indicating that the system has a significant synergistic bactericidal effect;

[0023] (4) The system exhibits higher encapsulation efficiency, better stability and excellent dispersibility.

[0024] The present invention also provides a method for preparing the above system, comprising: dissolving Surfactin in an aqueous phase and adjusting the pH to 7.0–8.0; preparing an oil phase containing a hydrophobic antibacterial active ingredient; adding the oil phase dropwise to the aqueous phase under stirring; subjecting the system to ultrasonic treatment or high-pressure homogenization to form a nanostructure; and obtaining a self-assembled nanosystem that meets the particle size, PDI and FIC indices.

[0025] The system provided by this invention exhibits significant synergistic antifungal activity. The mechanism is as follows: (1) Physical synergy: Surfactin reduces the interfacial tension of the fungal cell wall, promoting the rapid penetration of hydrophobic antibacterial active ingredient molecules into the cell. (2) Physiological synergy: The hydrophobic antibacterial active ingredient molecules entering the cell, together with Surfactin, synergistically disrupt the mitochondrial respiratory chain, leading to an exponential surge in intracellular reactive oxygen species levels (up to 5.8 times that of the control group), activating cysteine ​​proteases, and ultimately inducing DNA breakage and irreversible programmed cell death in fungal cells. The beneficial effect of this synergistic effect is that, compared to using either active ingredient alone, the system of this invention can reduce the amount of Surfactin used by 25%-60% and the amount of hydrophobic antibacterial active ingredient used by 30%-80%, thereby achieving high efficiency, enhanced efficacy with reduced dosage, and no chemical residue. The structure of the hydrophobic antibacterial active ingredient can be plant-derived terpenes / phenols (such as carvacrol and cinnamaldehyde), microbial-derived lipopeptides / antibiotics (such as echinocandin), or chemically synthesized hydrophobic triazoles, methoxyacrylates, and other bactericides.

[0026] This invention also provides the application of the above system in the control of filamentous fungi, which is applicable to various scenarios such as pre-harvest disease control and post-harvest preservation.

[0027] The innovative aspects of this invention include, but are not limited to:

[0028] 1. The criticality and unpredictability of a specific ratio (1:5-2:1)

[0029] This invention reveals for the first time that the system can only form a stable nanostructure and exhibit synergistic activity with FIC ≤ 0.5 within the A:B ratio range of 1:5 to 2:1. When the ratio deviates from this range, the particle size increases sharply, the PDI rises, FIC > 0.5, and the synergistic effect disappears, representing a nonlinear and unpredictable critical window. More importantly, this invention demonstrates through systematic FIC determination, ROS generation, TUNEL staining, and field trial data that the Surfactin nanosystem not only improves the physical dispersion of B but also produces a significant synergistic antibacterial effect with B (FIC ≤ 0.5), and induces significant ROS bursts and programmed cell death (PCD) characteristics in the target filamentous fungi. This synergistic effect and PCD induction were not observed in the Tween-80 system or other biosurfactant systems (such as rhamnolipid) targeting the same B, representing a typical unpredictable technical effect.

[0030] 2. The unique role of Surfactin in synergistic bactericidal action

[0031] Surfactin not only acts as an emulsifier, but also significantly improves the bioaccessibility of active ingredient B, making it a key factor in achieving synergistic effects; control systems such as Tween-80 and rhamnolipid did not exhibit similar synergistic effects.

[0032] 3. The nanosystem of this invention can induce fungal PCD.

[0033] The system of this invention can synergistically induce a sharp increase in ROS and activation of cysteine ​​proteases, ultimately inducing irreversible programmed cell death in fungal cells. This is a mechanism innovation that has never been achieved in traditional emulsification systems, further enhancing the bactericidal effect.

[0034] 4. High stability

[0035] The system exhibits a particle size change of less than 10% after 30 days of storage at room temperature, demonstrating significantly higher stability than other emulsifier systems and making it more suitable for industrial applications.

[0036] 5. Significantly reduces the amount of active ingredients used.

[0037] Compared to using any single active ingredient alone, the system of this invention can reduce the amount of Surfactin used by 25%-60% and the amount of hydrophobic antibacterial active ingredient used by 30%-80%, thereby achieving the goals of high efficiency, increased efficacy with reduced dosage, and no chemical residue.

[0038] 6. This invention reveals the mechanism of synergistic antifungal activity.

[0039] (1) Physical synergy (high permeability): Surfactin reduces the interfacial tension of the fungal cell wall, promoting the rapid penetration of hydrophobic antibacterial active ingredients into the cell. (2) Physiological synergy (induction of apoptosis): This is the key difference between this invention and existing technologies. The hydrophobic antibacterial active ingredients that enter the cell work synergistically with Surfactin: synergistically interfere with ion balance and synergistically disrupt the mitochondrial respiratory chain. The cascade amplification of the two leads to an exponential burst of intracellular reactive oxygen species (ROS) levels (up to 5.8 times that of the control group). The ROS burst further activates cysteine ​​proteases, leading to DNA fragmentation, and ultimately inducing irreversible programmed cell death in fungal cells.

[0040] In summary, the aforementioned proportional window, nanostructure stability, synergistic effect, and cascade relationship of inducing programmed cell death in fungi have not been revealed or reasonably derived in the prior art. This invention achieves a non-obvious technical breakthrough through holistic innovation of structure-proportion-mechanism. The technical solution of this invention has outstanding substantive features and significant progress, and possesses significant inventiveness.

[0041] The nano-drug delivery system described in this invention is based on an oil-in-water (O / W) liquid dispersant. Furthermore, this invention can further process it into solid dosage forms according to the needs of the agricultural and food industries. The liquid dispersant can be combined with known carriers (such as starch, kaolin, or silicates) and additives, and prepared into wettable powders, water-dispersible granules, or soluble powders through drying methods such as spray drying, fluidized bed granulation, or freeze drying. These solid dosage forms, after dilution with water, can still redisperse to form a stable nanosystem with an average particle size in the range of 20 nm to 200 nm, while maintaining their synergistic bactericidal activity.

[0042] The beneficial effects of this invention are as follows:

[0043] 1. Fully bio-derived emulsification and high stability: Eliminating traditional chemical emulsifiers, Surfactin, a natural lipopeptide, possesses an extremely low critical micelle concentration (CMC). It efficiently encapsulates hydrophobic antibacterial active ingredients into uniformly sized, long-term stable (PDI < 0.3, no stratification at room temperature > 30 days) nanodroplets, significantly overcoming the shortcomings of poor water solubility, volatility, or low stability of hydrophobic antibacterial ingredients.

[0044] 2. Unexpected Strong Synergistic Effect (FIC≤0.5): This invention reveals that at specific A:B ratios of 1:5 to 2:1, the FIC index of the system significantly decreases to below 0.5. Comparative experiments (Example 10) demonstrate that systems constructed using Tween-80 or rhamnolipids under the same conditions have FICs of 1.25 and 0.76, respectively, neither exhibiting a synergistic effect. This proves that the synergistic effect of this invention is unique to Surfactin, rather than a common property of ordinary surfactants.

[0045] 3. Breakthrough Enhancement and Reduced Dosage: Thanks to the aforementioned strong synergistic mechanism, this system achieves a significant reduction in the amount of active ingredients used. Field trials show that even with reductions of 76% and 80% in the dosage of Surfactin and propiconazole, respectively, the field efficacy (85.0%) is still significantly superior to that of the commercially available chemical pesticide tebuconazole (74.9%) when applied in full. This solves the long-standing dual problems of high cost of biopesticides and high residues of chemical pesticides.

[0046] 4. Excellent physical stability: Utilizing the unique cyclic lipopeptide structure of Surfactin, the nanoparticles formed within a limited ratio exhibit excellent monodispersity (PDI≤0.3), with a particle size change of <10% after 30 days of storage at room temperature, which is significantly better than the broad distribution characteristics of the rhamnoglycolipid system (PDI=0.41).

[0047] 5. Broad Spectrum Applicability: This nano-drug delivery system is not only applicable to plant essential oils (such as carvacrol and cinnamaldehyde), but also to a variety of microbial antibiotics (such as echinocandin and fenestrant) and chemically synthesized hydrophobic bactericides (such as triazoles and methoxyacrylates, which can all be efficiently encapsulated and synergistically enhanced by Surfactin based on their hydrophobic structural characteristics), significantly expanding its application scope and technological value.

[0048] Comparative analysis of existing technologies

[0049] This invention selects Surfactin as active ingredient A, which is fundamentally different from commonly used chemically synthesized surfactants (such as Tween-80) in the prior art, strongly supporting the inventiveness of this invention. Chemically synthesized surfactants and other biosurfactants only serve as inert excipients with limited functions, mainly responsible for physical emulsification and system stabilization, and do not participate in the bactericidal process; their effects are predictable. However, Surfactin, as a unique cyclic lipopeptide biosurfactant, achieves a non-obvious function of "two uses in one": it not only possesses highly efficient emulsification and self-assembly capabilities, but more importantly, it is also a synergistic bactericidal active ingredient. Studies show that Surfactin can actively promote the penetration of hydrophobic active ingredients into fungal cell membranes and synergistically induce an exponential burst of intracellular reactive oxygen species (ROS), thereby producing a strong synergistic effect with FIC ≤ 0.5. It is this proactive synergistic mechanism that enables the system of this invention to achieve field efficacy superior to commonly used chemical control agents (e.g., 85.0% vs 74.9%) even with a significant reduction in the amount of active ingredients (more than 70%), thus solving the problem of balancing efficacy enhancement and dosage reduction with green environmental protection that cannot be achieved by existing technologies.

[0050] Furthermore, the choice of Surfactin in this invention is not a simple replacement for other biosurfactants in the prior art (such as rhamnolipids and sophorolipids), but rather based on the irreplaceable technical effects conferred by its unique cyclic lipopeptide structure. Compared to other biosurfactants, Surfactin has superior surface activity and a lower critical micelle concentration (CMC), enabling it to achieve efficient encapsulation and stable self-assembly of hydrophobic active ingredients at extremely low concentrations. More importantly, experimental data from this invention demonstrate that only Surfactin can produce a specific synergistic effect with hydrophobic active ingredients, including stronger promotion of active ingredient transmembrane penetration and specific participation in intracellular ROS signaling and apoptosis pathways. This synergistic effect is not common to other biosurfactants, and a strong synergistic effect with FIC ≤ 0.5 can only be stably achieved within the critical mass ratio range of 1:5 to 2:1 defined in this invention, ultimately supporting the unexpected results of synergistic effect with reduced dosage and surpassing the efficacy of chemical pesticides.

[0051] In summary, this invention differs fundamentally from the simple emulsifier-drug compounding in known technologies. In known technologies, the emulsifier merely acts as an inert excipient, and its dosage has no direct relationship with efficacy. This invention creatively discovers that Surfactin is not only a carrier but also a synergistic active ingredient. As shown in Example 9, when the mass ratio of Surfactin to the hydrophobic active ingredient falls within the critical range of 1:5 to 2:1, they can achieve a strong synergistic effect with FIC ≤ 0.5, simultaneously forming nanoparticles with stable particle size and no long-term stratification. When the ratio exceeds this range (e.g., 1:6 or 3:1), the synergistic effect disappears (FIC > 0.5), or the physical stability of the system deteriorates sharply. Furthermore, this invention reveals that at doses far below its inhibitory concentration (MIC), Surfactin induces programmed cell death, exponentially increasing the bactericidal efficacy of the hydrophobic drug, ultimately achieving an unexpected technical effect of reducing the total amount of active ingredient by more than 70% while still maintaining higher field efficacy than the chemical control agent. This efficiency improvement and quantity reduction achieved based on a specific ratio and a specific synergistic mechanism is the most creative and substantial feature of this invention.

[0052] This invention provides a groundbreaking technical solution based on in-depth research into the collaborative mechanism. Attached Figure Description

[0053] Figure 1 Schematic diagram of Surfactin and its self-assembled nanoparticle structure Detailed Implementation

[0054] Example 1: Preparation of Surfactin-Carvacrol Nanoemulsion

[0055] 50 mg of 95% pure Surfactin sodium salt was weighed and dissolved in 10 mL of PBS buffer (pH 7.4) as the aqueous phase. 100 mg of carvacrol was measured as the oil phase. Under magnetic stirring, carvacrol was added dropwise to the Surfactin solution, followed by ultrasonic treatment with a probe under ice bath conditions (400 W power, 3 s operation, 3 s interval, for a total of 10 minutes). The resulting nanoemulsion was a clear, transparent, pale yellow liquid.

[0056] The average particle size and polydispersity index (PDI) were determined using dynamic light scattering (DLS). The nanoparticle-loaded drug system was diluted with ultrapure water at a volume ratio of 1:100, equilibrated at 25℃±2℃ for 5 min, and then measured using an instrument such as the Malvern Zetasizer. Three measurements were taken, and the average was recorded. The Z-mean particle size and PDI were reported. The Z-mean particle size is used to assess the size of the nanoparticles, and the PDI is used to assess the uniformity of the particle size distribution. A PDI value less than or equal to 0.3 indicates that the system has good monodispersity and stability.

[0057] The test results showed that the average particle size was 45 nm, the polydispersity index (PDI) was less than 0.2, and no stratification or demulsification occurred after 30 days of storage at room temperature, indicating excellent stability.

[0058] Example 2: Preparation of Surfactin-Echinocandin Nanoparticle Drug Delivery System

[0059] 60 mg of 95% pure Surfactin sodium salt was weighed and dissolved in 10 mL of Tris-HCl buffer (pH 7.5) as the aqueous phase. 80 mg of echinocandin (a microbial hydrophobic antibiotic) was measured as the oil phase. Under magnetic stirring, echinocandin was added dropwise to the Surfactin solution, followed by five cycles of high-pressure homogenization at 800 bar. The resulting nanoparticle-loaded drug system appeared as a translucent milky-white liquid. Dynamic light scattering (DLS) analysis showed an average particle size of 85 nm, a polydispersity index (PDI) of less than 0.25, and no stratification or demulsification occurred after 30 days at room temperature, indicating good stability.

[0060] Example 3: Preparation of Surfactin-propiconazole nanocarrier system

[0061] 70 mg of 95% pure Surfactin sodium salt was weighed and dissolved in 10 mL of deionized water. The pH was adjusted to 7.8 with NaOH solution to form the aqueous phase. 70 mg of the chemically synthesized hydrophobic bactericide propiconazole was measured to form the oil phase. Under magnetic stirring, propiconazole was added dropwise to the Surfactin solution, followed by ultrasonic treatment with a probe under ice bath conditions (350 W power, 5 s operation, 5 s interval, 8 minutes total). The resulting nano-drug-loaded system was a semi-transparent liquid. Dynamic light scattering (DLS) analysis showed an average particle size of 120 nm, a polydispersity index (PDI) of less than 0.3, and no obvious stratification or precipitation occurred after 30 days at room temperature, indicating stability meeting application requirements.

[0062] Example 4: Stability Analysis of Nanoparticle Drug Delivery System

[0063] To comprehensively evaluate the stability of the nano-drug delivery system of this invention, we performed dynamic light scattering (DLS) analysis on three typical systems after preparation and examined their changes after storage at room temperature (25℃±2℃) for 30 days.

[0064] Table 1. Stability of different nano-drug delivery systems

[0065]

[0066] The above results indicate that whether it is plant essential oil, microbial antibiotics, or chemically synthesized fungicides, the self-assembled nano-drug delivery system constructed based on Surfactin in the present invention can maintain excellent physical stability at small particle sizes (between 20 - 200 nm). In particular, the PDI values of all systems are less than 0.3, showing a narrow and uniform particle size distribution. This high stability benefits from the extremely low critical micelle concentration (CMC) and excellent amphiphilicity of Surfactin, avoiding problems such as Ostwald ripening or coalescence and delamination that are prone to occur in traditional emulsions. The high stability of the nano-drug delivery system also lays a key material foundation for the exertion of drug efficacy and the stability of control efficacy.

[0067] Example 5: Determination of Synergistic Bacteriostatic Activity (FIC Index Analysis)

[0068] To clearly prove that the synergistic effect of the nano-drug delivery system of the present invention is not a simple superposition of active ingredients, we conducted a comparative analysis of the dosages of active ingredients in each system.

[0069] The minimum inhibitory concentration (MIC) was determined by the microbroth dilution method. That is, a fungal suspension with a concentration of 1×10 5 spores / mL was prepared, and it was mixed and incubated with a series of serially diluted single-agent active ingredient solutions (or drug delivery systems) in a 96-well plate for 48 hours; by visual observation or measuring the absorbance at 600 nm, the lowest drug concentration at which no visible mycelial growth appeared was defined as the MIC value of the active ingredient.

[0070] The synergistic effect index (FIC) was determined by the checkerboard dilution method. That is, a cross-dilution matrix of active ingredient A (Surfactin) and active ingredient B was designed in a 96-well plate, and an equal volume of fungal suspension was added and then cultured; according to the lowest combined concentration combination that completely inhibited fungal growth, the FIC index was calculated as MIC A+B / MIC A +(MIC B+A / MIC B , where FIC ≤ 0.5 is synergistic, 0.5 < FIC ≤ 1 is additive, 1 < FIC ≤ 4 is irrelevant, and FIC > 4 is antagonistic. All experiments were repeated 3 times, with 3 parallels set each time.

[0071] Table 2 Synergistic Bacteriostatic Effects of Different Nano-Drug Delivery Systems

[0072]

[0073] As shown in the table above, both Surfactin and the hydrophobic antibacterial active ingredient require high minimum inhibitory concentrations (MICs) when used alone. However, when the two are combined in the nanosystem form described in this invention, not only are the FIC indices less than 0.5, but the actual usage concentrations (MICs after combination) of the two active ingredients are also significantly reduced by 70% to 83.3% compared to their individual MICs.

[0074] This significant reduction in dosage and enhanced antibacterial effect far exceeded expectations, strongly demonstrating that Surfactin is not merely a simple emulsifier, but rather produces an unexpected synergistic effect through physical synergy (aided penetration) and physiological synergy (induction of apoptosis). Therefore, the nano-drug delivery system of this invention possesses outstanding inventiveness.

[0075] Example 6: Validation of the mechanism of induced programmed cell death

[0076] This embodiment aims to verify, through cytological testing, that the Surfactin nanocarrier system described in this invention (taking the Surfactin-carvacrol system as an example) kills filamentous fungi through programmed cell death rather than simple necrosis.

[0077] 1. Mycelial treatment and grouping

[0078] Fresh Aspergillus mycelia were collected and divided into four groups: a blank control group (CK, culture medium only), a single-agent Surfactin group, a single-agent hydrophobic antibacterial active ingredient group (carvacrol), and a treatment group using the nano-drug delivery system of this invention (using a combined inhibitory concentration of FIC ≤ 0.5). All treatment groups were incubated for 4 hours, after which mycelia were collected for subsequent testing.

[0079] 2. Mechanism Validation Methods and Results

[0080] (1) Detection of reactive oxygen species accumulation

[0081] Intracellular reactive oxygen species (ROS) accumulation was detected using the DCFH-DA fluorescent probe method. Treated hyphae were collected, washed with PBS buffer, and then immersed in 10 μM DCFH-DA probe solution, incubated at 37°C in the dark for 30 minutes. Intracellular ROS oxidize the non-fluorescent DCFH to strongly fluorescent green DCF. After washing, fluorescence detection was performed using a laser confocal microscope or a fluorescence microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. All quantitative fluorescence assays were repeated three times, and statistical tests were performed (p < 0.05).

[0082] The results showed that Aspergillus flavus mycelial cells treated with the nano-drug delivery system of this invention exhibited strong green fluorescence (ROS burst). Quantitative analysis showed that the fluorescence intensity of this treatment group was 5.8 times that of the control group and significantly higher than that of the groups treated with Surfactin or carvacrol alone, confirming that the synergistic effect triggered a sharp increase in intracellular oxidative stress.

[0083] (2) Detection of mitochondrial membrane potential decrease

[0084] The decrease in mitochondrial membrane potential was detected using the JC-1 staining method. Treated hyphae were collected, washed with PBS buffer, and then incubated with 10 μg / mL JC-1 dye solution for 20 minutes in the dark. In healthy cells, JC-1 accumulated in the mitochondria as a red fluorescent polymer; in apoptotic cells, the membrane potential collapsed, and JC-1 diffused in the cytoplasm as a green fluorescent monomer. Results: Control group hyphae showed strong red fluorescence, while the mitochondria of hyphae treated with the nanoparticle-loaded drug system showed significant fading, changing from red fluorescence to green fluorescence (the red / green fluorescence ratio decreased significantly), indicating mitochondrial membrane potential collapse, an early marker of programmed cell death.

[0085] (3) Detection of Caspase-like protease activity activation

[0086] Caspase-like protease activation was detected using the FITC-VAD-FMK probe method. After treatment, mycelia were collected, washed with PBS, and then the FITC-labeled pan-cysteine ​​inhibitor probe FITC-VAD-FMK was added. The cells were incubated at 37°C in the dark for 30 minutes to allow the probe to covalently bind to the activated caspase-like protease. After washing, the cells were detected using laser confocal fluorescence microscopy. Increased fluorescence signal indicated caspase-like protease activation. Results: The FITC fluorescence intensity of mycelial cells treated with the nanoparticle-loaded drug system of this invention was significantly higher than that of the control group, indicating that the key apoptosis effector protease (caspase-like protease) had been activated. This provides direct molecular evidence for determining that fungal death is programmed cell death.

[0087] (4) DNA breakage detection

[0088] Nuclear DNA fragmentation was detected using a TUNEL assay kit. Fixed hyphae were permeabilized and incubated with a mixture of TdT enzyme and dUTP fluorescently labeled molecules. In apoptotic cells, the broken DNA ends were labeled with fluorescent signals. Results: Microscopic observation and quantitative analysis showed that approximately 85% of the cell nuclei in the group treated with the nanoparticle-loaded drug system exhibited TUNEL-positive fluorescence signals, confirming intranuclear DNA fragmentation, consistent with typical late-stage characteristics of programmed cell death.

[0089] Based on the above cytological test results, the fungal death induced by the Surfactin nanoparticle drug delivery system exhibited typical characteristics of programmed cell death, such as ROS burst, decreased mitochondrial membrane potential, activation of Caspase-like protease, and DNA breakage. This strongly confirms that the system of this invention exerts a synergistic bactericidal effect through a synergistic apoptosis-inducing mechanism.

[0090] Example 7: Application and quality evaluation in postharvest preservation of strawberries

[0091] Postharvest mold is the main cause of strawberry losses, and the culprits are a series of fungi. Among them, gray mold caused by Botrytis cinerea is the most common postharvest disease, while other common molds include Rhizopus, Penicillium, and Mucor.

[0092] Fresh strawberries (variety "Hongyan") were randomly divided into three groups: (1) blank control group (CK, treated with water); (2) ordinary control group (Tween-80 emulsion containing 100 μg / mL carvacrol); (3) treatment group 1 of this invention (Surfactin nano-drug delivery system containing 100 μg / mL carvacrol); (4) treatment group 2 of this invention (Surfactin nano-drug delivery system containing 50 μg / mL propiconazole). The treatment time was 30 seconds for all groups, and after air drying, the strawberries were stored at 25℃ for 8 days.

[0093] The results are shown in Table 2. After 8 days of storage, the rot rate of strawberries in the treatment group of this invention was significantly lower than that in the control group (p≤0.05). Particularly noteworthy is that the Surfactin-propiconazole nano-loaded drug system achieved a preservation effect similar to the Surfactin-carvacrol system using only half the concentration of the active ingredient, demonstrating the wide applicability and high efficiency of this nano-loaded drug system. This invention's nano-loaded drug system not only more effectively kills latent fungi on the fruit surface, but also, due to Surfactin's excellent film-forming properties, forms a semi-permeable biological membrane on the fruit surface, significantly reducing weight loss (p≤0.05) and delaying the decrease in fruit firmness. This proves that this nano-system has dual effects of "bactericidal" and "film-forming preservation" in the preservation of fruits and vegetables.

[0094] Table 3. Effects of different treatments on the decay rate and physicochemical properties of strawberries during storage.

[0095]

[0096] Example 8: Field control efficacy of Surfactin-propiconazole nanocarrier system against wheat scab.

[0097] Experimental location: A wheat scab disease-prone plot in Huai'an, Jiangsu Province. A randomized block design was used, with 3-4 replicates per treatment. Each plot was 20 m².2 .

[0098] Test reagents: Blank control (CK): Water (with a small amount of adjuvant, without active ingredient). Surfactin single agent: 50 μg / mL Surfactin aqueous solution (set at the original single-agent MIC high dose). Propiconazole single agent: 50 μg / mL Propiconazole EC (set at the original single-agent MIC dose). Common chemical pesticide control (PC): Tebuconazole (a commonly used triazole fungicide, set at the conventional recommended concentration of 50 μg / mL). Example 3 prepared the Surfactin-propiconazole nanocarrier system (Surfactin:propiconazole mass ratio approximately 1:1, total active ingredient concentration adjusted to 24 μg / mL according to FIC results, of which propiconazole concentration was 12 μg / mL).

[0099] Application method: Apply the pesticide evenly by spraying during the wheat flowering stage (the critical period for Fusarium head blight infection). Repeat the application once after 3 days. Data measurement: 15 days after application, investigate the disease condition according to standard methods, and calculate the disease index and field control efficacy for each treatment.

[0100] The results are shown in Table 4. (1) Significant synergistic effect: The efficacy of single-agent Surfactin was only 10.0%, and the efficacy of single-agent propiconazole was 40.0%. If the two were simply added together, the expected efficacy would theoretically not exceed 50%. However, the Surfactin-propiconazole nano-loaded system of the present invention achieved a field efficacy of up to 85.0% when only a low dose of active ingredient was used. This is much higher than the efficacy when the two are used alone, which strongly proves the actual effect of synergistic effect in the field environment. (2) Significant effect enhancement and dosage reduction: The dosage of propiconazole in group E of the system of the present invention (12ug / mL) is much lower than that in group C (50ug / mL) and group D (50ug / mL) of commonly used chemical pesticides. Even though the total amount of active ingredient decreased by about 70%, the final effect was still much higher than that of the commercial chemical fungicide tebuconazole (74.9%), which is obviously an unexpected result.

[0101] Table 4 Field control efficacy against wheat scab

[0102]

[0103] Example 9: The effect of mass ratio on system stability and synergistic effect

[0104] To demonstrate that the optimal mass ratio of Surfactin to the hydrophobic antibacterial active ingredient, ranging from 1:5 to 2:1, is a necessary limitation for achieving the technical effects of this invention, we used the Surfactin-carvacrol system as an example to investigate the stability and synergistic antibacterial effect of systems outside this range.

[0105] Experimental method: Keeping the total active ingredient concentration constant, the mass ratio of Surfactin (A) to carvacrol (B) was changed, and the system was prepared according to the method of Example 1. The appearance stability (observe layering or demulsification within 4 hours) and FIC index were measured.

[0106] Table 5. Effect of mass ratio on the stability and synergistic effect of nano-drug delivery systems

[0107]

[0108] The results showed that when the surfactin content was too low (A:B < 1:5), surfactin could not provide sufficient interfacial coverage and electrostatic repulsion, resulting in unstable dispersion of the hydrophobic antibacterial components. The system underwent macroscopic stratification and demulsification within a short time, failing to form a stable nano-drug-carrying system, thus rendering the stability ineffective and the preventative efficacy meaningless. When the surfactin content was too high (A:B > 2:1), although the system was stable, excessive surfactin would occupy key target sites on the fungal cell membrane through competitive adsorption or saturation, inhibiting the synergistic effect of the hydrophobic antibacterial components, resulting in an FIC index greater than 0.5. This indicates that the combined effect at this point is no longer the strong synergistic effect required by this invention. These experimental results demonstrate that the mass ratio of 1:5 to 2:1 specified in claim 1 is the critical technical range for achieving the stable nano-system and strong synergistic effect (FIC ≤ 0.5) of this invention. This provides indispensable evidence for the non-obviousness of this core parameter.

[0109] Example 10: Comparative Analysis of Nanoparticle Drug Delivery Systems Constructed with Different Surfactants

[0110] To highlight the non-obviousness of the active ingredient A selected in this invention in the construction of the nano-drug delivery system and synergistic effect, this embodiment selects Tween-80, a commonly used chemical surfactant in the prior art, and another common biosurfactant, rhamnolipid, as controls for comparative experiments with the Surfactin system of this invention.

[0111] 1. Experimental materials:

[0112] Active ingredient B: carvacrol; chemically synthesized surfactant: Tween-80; biosurfactant: rhamnolipid. Test subject: Fusarium graminearum.

[0113] 2. System preparation and characterization

[0114] All systems were prepared according to the method described in Example 1, with the mass ratio of surfactant to carvacrol consistently set at 1:1. After preparation, the nanostructure stability (average particle size and PDI) and synergistic effect (FIC value) of the systems were measured.

[0115] 3. Results and Analysis

[0116] Table 6 Comparative analysis of nano-drug delivery systems constructed with different surfactants

[0117]

[0118] (1) Comparative Analysis of Nanostructure Formation and Stability: Control Group B: Tween-80 is a well-known nonionic emulsifier that can form nanoscale particles through physical encapsulation under appropriate energy. Its PDI value of 0.29 verifies that the system has basic stability. However, as a long-chain polyoxyethylene sorbitan ester, its structure lacks the strong compact packing property brought by the cyclic structure of Surfactin, making it difficult for the nanostructure formed to achieve the best monodispersity and long-term kinetic stability shown by the system of this invention with PDI = 0.24. Control Group C: As another biosurfactant, rhamnolipids have different molecular geometry packing requirements due to their glycolipid structure compared to the cyclic lipopeptide structure of Surfactin. This structure results in lower packaging density and higher interfacial curvature stress at the oil / water interface, making it difficult to efficiently and stably encapsulate hydrophobic components. The experimental result of PDI = 0.41 (higher than 0.3) directly proves that the rhamnolipid system has a wide particle size distribution and poor monodispersity, making it difficult to meet the requirements of this invention for a stable nano-drug-carrying system.

[0119] (2) Comparative Analysis of Synergistic Effects: Control Group B: Tween-80 is biologically inert, and its only function is to increase the solubility of active ingredient B in the aqueous phase. Its FIC = 1.25 indicates that this simple physical dissolution effect cannot trigger synergistic effects, and may even slightly interfere with the efficacy of active ingredient B by occupying potential binding sites on the cell membrane (FIC > 1.0). Control Group C: Although rhamnolipid has basic antibacterial activity, its structure determines that it lacks the same specific biological function as Surfactin. It cannot effectively and specifically trigger the ROS burst signaling pathway and programmed cell death mechanism in fungal cells. Therefore, its FIC = 1.75 indicates that the combination of rhamnolipid and carvacrol failed to produce a structural or functional synergistic effect, which is fundamentally different from the synergistic effect result of FIC ≤ 0.5 in this invention.

[0120] In summary, the Surfactin self-assembled nanoparticle drug delivery system provided by this invention is based on its unique cyclic lipopeptide structure, which simultaneously achieves optimal nanostructure construction (low PDI) and specific biosynergistic effects (low FIC). This is not possessed by other chemical or biological surfactants, and therefore this invention represents a non-obvious technological breakthrough.

[0121] Example 11: Preparation and Synergistic Analysis of a Nanoparticle Drug Delivery System for Surfactin and a Microbial Antibiotic (Echinocandin)

[0122] In this embodiment, the microbial antibiotic echinocandin was selected as the hydrophobic antibacterial active ingredient B, and the mass ratio of Surfactin to echinocandin was set at 1:5. The system was prepared using a high-pressure homogenization method. Dynamic light scattering (DLS) analysis showed an average particle size of 118.9 ± 6.3 nm and a PDI of 0.27 ± 0.04, demonstrating its ability to stably form a uniform nanostructure, meeting the stability requirements of this invention. The checkerboard dilution method was used to determine the antibacterial synergy of this system against *Botrytis cinerea*. The results showed an FIC of 0.45, which is lower than 0.5, proving that Surfactin can produce a strong synergistic effect with microbial hydrophobic antibacterial components of different structures.

[0123] Example 12 Preparation and Synergistic Analysis of a Nanoparticle Drug Delivery System of Surfactin and Plant Essential Oil (Cinnamaldehyde)

[0124] In this embodiment, cinnamaldehyde, a plant essential oil, was selected as the hydrophobic antibacterial active ingredient B, and the mass ratio of Surfactin to cinnamaldehyde was set at 1:2. The system was prepared using ultrasonic dispersion, and the average particle size was measured by DLS, with a PDI of 78.3 ± 3.5 nm and a PDI of 0.22 ± 0.02, demonstrating excellent stability and homogeneity, proving the applicability of this system to various essential oils. The antibacterial synergistic effect against Fusarium oxysporum was determined using the checkerboard dilution method. The results showed a synergistic enhancement index (FIC) of 0.35, consistent with previous examples regarding carvacrol, further verifying the universality of the synergistic mechanism of this invention.

[0125] For those skilled in the art, modifications can still be made to the technical solutions described in the above embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by this invention.

Claims

1. A self-assembled nano-synergistic antibacterial system constructed based on Surfactin, characterized in that: The system is an oil-in-water (O / W) structure with water as the continuous phase, comprising: active ingredient A is Surfactin or its salt, as an amphiphilic assembly framework and synergist; active ingredient B is a hydrophobic antibacterial active ingredient selected from one or any proportion combination of plant essential oils, microbial source antibiotics or chemically synthesized hydrophobic fungicides; wherein the mass ratio (A:B) of active ingredient A to active ingredient B is 1:5 to 2:1, within the mass ratio range, active ingredient A and active ingredient B form stable nanoparticles through self-assembly, and produce synergistic antibacterial effect, and meet the following characteristics: (1) the average particle size of the nanoparticles is 20-200 nm, (2) the polydispersity index (PDI) is ≤0.3, (3) the synergistic index (FIC) is ≤0.5, (4) active ingredient A and active ingredient B synergistically induce target fungal mitochondrial membrane potential collapse and intracellular reactive oxygen species (ROS) burst, triggering programmed cell death.

2. The nanosyrnergic antibacterial system according to claim 1, characterized in that: The Surfactin is a cyclic lipopeptide obtained by fermentation of Bacillus and purified, with a purity of ≥95%, and the concentration of Surfactin in the system is lower than the minimum inhibitory concentration (MIC) when used alone, and the bactericidal activity is exerted through synergistic effect.

3. The nanosyrnergic antibacterial system according to claim 1, characterized in that: The plant essential oil is selected from one or a combination of carvacrol, thymol, eugenol, isoeugenol, nerol, neral, citral, cinnamaldehyde or vanillin.

4. The nanosyrnergic antibacterial system according to claim 1, characterized in that: The microbial source antibiotic is a hydrophobic antibiotic that has a synergistic relationship with Surfactin, selected from one or a combination of echinocandins (such as caspofungin, micafungin), fengycin or iturin; preferably, the fengycin or iturin is a purified product with a purity of ≥90%, and is artificially compounded with Surfactin according to the mass ratio.

5. The nanosyrnergic antibacterial system according to claim 1, characterized in that: The chemically synthesized hydrophobic fungicide is selected from one or a combination of triazoles, methoxy acrylates or succinate dehydrogenase inhibitors (SDHIs); preferably selected from propiconazole, tebuconazole, flusilazole, difenoconazole, hexaconazole (triazoles), azoxystrobin, pyraclostrobin (methoxy acrylates) or boscalid (SDHIs).

6. A method of preparing the nano-synergistic antibacterial system of claim 1, characterized in that, The method comprises the following steps: (1) dissolving active ingredient A in buffer or water, adjusting the pH to 7.0-8.0 to obtain an aqueous phase; (2) adding active ingredient B as an oil phase to the aqueous phase under stirring; (3) treating with ultrasonic crushing (power 300-500 W, processing time 5-15 minutes) or high-pressure homogenization (pressure 500-1000 bar, cycle 3-10 times), utilizing the low critical micelle concentration (CMC) characteristics and amphiphilic structure of Surfactin, to form a stable self-assembly nanosystem with a particle size of 20-200 nm, PDI ≤0.3 and FIC ≤0.

5.

7. Use of the nano-synergistic antibacterial system of claim 1 in the preparation of biological pesticides or food preservatives for the prevention and control of filamentous fungi.

8. Use of the nanosynergetic antimicrobial system according to claim 1 for the preparation of a liquid dispersion, a wettable powder, a water dispersible granule or a soluble powder, characterized in that, The solid dosage form can be re-dispersed to form stable nano with average particle size of 20-200nm after adding water.

9. Use according to claim 7, characterized in that: The filamentous fungi include, but are not limited to, Fusarium graminearum, Aspergillus flavus, Botrytis cinerea, Fusarium oxysporum, Penicillium expansum.

10. Use according to claim 7, characterized in that, The prevention and control mechanism includes that surfactin reduces the interfacial tension of fungal cells, promotes the entry of active ingredient B into the fungal cells, surfactin and active ingredient B synergistically destroy the mitochondrial function, induce intracellular active oxygen burst, activate caspase and cause DNA fragmentation, thereby inducing fungal programmed cell death.

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