Rainwater-washing-resistant and long-acting antibacterial liquid-liquid condensed phase as well as preparation method and application thereof

Abstract

The invention provides a rain wash resistant and long-acting antibacterial liquid-liquid condensed phase and a preparation method and application thereof.The liquid-liquid condensed phase comprises polyethyleneimine, sodium deoxycholate and metal chloride salt, the concentration of the polyethyleneimine is 0.5 g / L-5 g / L, and the molecular weight of the polyethyleneimine is 600-25000; the concentration of the sodium deoxycholate is 0.5 mM to 50 mM.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the technical field of pesticides, in particular to a rain-resistant and long-acting antibacterial liquid-liquid coacervate as well as a preparation method and application thereof, and more particularly to a liquid-liquid coacervate, a preparation method thereof, a rain-resistant bactericide and application of the rain-resistant bactericide in agricultural sterilization. BACKGROUND

[0002] In agricultural production, agricultural bactericides play an important role in preventing and controlling plant diseases. However, the utilization efficiency and long-acting property of the agricultural bactericides are significantly affected by rainfall. On the one hand, rainfall promotes the spread of plant pathogens, leading to frequent outbreaks of diseases in the rainy season; on the other hand, rainfall also washes away the agricultural bactericides that have not been absorbed by plants, thereby reducing the utilization rate of the agricultural bactericides. Especially for agricultural bactericides with high polarity and good water solubility, repeated application is often required in the rainy season, which not only increases the cost of agricultural production, but also increases the risk of agricultural non-point source pollution.

[0003] At present, various methods have been explored to enhance the leaf adhesion and rain resistance of agricultural bactericides, such as utilizing the size effect of nano-agricultural bactericides, adding adhesion promoters, modifying affinity groups, and improving the flexibility of the system, to enhance the leaf adhesion and rain resistance of agricultural bactericides. However, there are still problems such as complex synthesis and modification, high cost, and poor biocompatibility of some materials, which are not conducive to large-scale promotion.

[0004] Therefore, it has become an important direction for the development of green long-acting agricultural bactericides to develop a rain-resistant antibacterial system with simple preparation process, low toxicity and high efficacy. SUMMARY

[0005] Therefore, the main purpose of the present disclosure is to provide a rain-resistant and long-acting antibacterial liquid-liquid coacervate as well as a preparation method and application thereof, in order to at least partially solve at least one of the above-mentioned technical problems.

[0006] To achieve the above-mentioned purposes, the technical solutions of the present disclosure are as follows:

[0007] In one aspect of the present disclosure, a liquid-liquid coacervate is provided, comprising: polyethyleneimine and sodium deoxycholate, wherein the concentration of the polyethyleneimine is 0.5 g / L to 5 g / L, and the molecular weight is 600 to 25000; the concentration of the sodium deoxycholate is 0.5 mM to 50 mM.

[0008] In a second aspect of the present disclosure, a preparation method of the above-mentioned liquid-liquid coacervate is provided, comprising:

[0009] dissolving the polyethyleneimine in water and adjusting the pH to 6 to 8 to obtain a polyethyleneimine mother liquor;

[0010] mixing the polyethyleneimine mother liquor with sodium deoxycholate and a metal chloride salt, and stirring to obtain the liquid-liquid coacervate.

[0011] According to a third aspect of the present disclosure, there is provided a rain-resistant fungicide comprising the liquid-liquid coacervate or obtained by the preparation method.

[0012] According to a fourth aspect of the present disclosure, there is provided a use of the rain-resistant fungicide in agricultural fungicide.

[0013] According to embodiments of the present disclosure, the full water-based liquid-liquid coacervate spontaneously formed by sodium deoxycholate and polyethyleneimine through electrostatic and hydrophobic interactions has the advantages of low biological toxicity, environmental friendliness and biological safety. The network microstructure constructed by the system endows the system with good encapsulation performance, flexibility and wet surface adhesion, which can significantly improve the rain erosion resistance. In addition, the sodium deoxycholate destroys the fungal cell membrane, and the cationic antibacterial properties of polyethyleneimine form a synergistic effect, so that the liquid-liquid coacervate has broad-spectrum antibacterial activity and can exhibit outstanding application value in the field of agricultural fungicide. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Turbidity curves of polyethyleneimine and sodium chloride mixed solutions with different molecular weights with increasing sodium deoxycholate concentration;

[0015] Figure 2 A microscope image of the liquid-liquid coacervate in Example 1 of the present disclosure;

[0016] Figure 3 A confocal fluorescence image of the liquid-liquid coacervate in Example 1 of the present disclosure after encapsulating different fluorescent dyes;

[0017] Figure 4 A scanning electron microscope image of the liquid-liquid coacervate in Example 2 of the present disclosure;

[0018] Figure 5a A schematic diagram of a simulated rain washing system;

[0019] Figure 5b Leaf fluorescence images of the liquid-liquid coacervate and commercial adjuvants encapsulating rhodamine 6G in Example 2-3 of the present disclosure after simulated rain washing on the surface of lotus leaves;

[0020] Figure 5c Leaf fluorescence images of the liquid-liquid coacervate and plant essential oil adjuvants encapsulating rhodamine 6G in Example 2-3 of the present disclosure after simulated rain washing for different lengths of time on the surfaces of rice, wheat and cucumber leaves;

[0021] Figure 6 Scanning electron microscope images of the liquid-liquid coacervate in Example 2 of the present disclosure before and after spraying on the surfaces of lotus leaves, rice, wheat and cucumber leaves;

[0022] Figure 7A schematic diagram of an adhesion test system;

[0023] Figure 8 A diagram of an adhesion test of a liquid-liquid coacervate and water in Example 2 of the present disclosure;

[0024] Figure 9 A plot of adhesion change of a liquid-liquid coacervate and a polyethyleneimine solution and water in Examples 2-5 of the present disclosure;

[0025] Figure 10 A plot of the inhibition rate of a liquid-liquid coacervate, sodium deoxycholate, polyethyleneimine, a silicone adjuvant, hydroxylalkynyl-terminated dimethylpolysiloxane, and a polyether-modified heptamethyltrisiloxane of Examples 2-3 of the present disclosure on Rhynchosporium oryzae;

[0026] Figure 11 A plot of the inhibition rate of a liquid-liquid coacervate, sodium deoxycholate, polyethyleneimine, and sodium chloride on 11 common plant pathogenic fungi of Examples 2-3 of the present disclosure;

[0027] Figure 12 A plot of the antibacterial test effect of a liquid-liquid coacervate of Examples 2 and 4 of the present disclosure on Rhizoctonia solani after rainwater flushing on the surface of rice leaves;

[0028] Figure 13 A plot of the germination rate, root length, and shoot length of wheat seeds cultured with a dilute solution of a liquid-liquid coacervate and a silicone adjuvant, polyether-modified heptamethyltrisiloxane, and hydroxylalkynyl-terminated dimethylpolysiloxane of Examples 2-3 of the present disclosure. DETAILED DESCRIPTION

[0029] In order to make the objectives, technical solutions, and advantages of the present disclosure clearer, the present disclosure will be further described in detail below with specific examples.

[0030] The endpoints of the ranges and any values disclosed in the present disclosure are not limited to the precise values stated. The ranges or values should be construed to be approximations that allow for significant variation. Various

[0031] The terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting of the present disclosure. As used herein, the term "includes" and tautological equivalents thereof, means that the claimed features, steps, operations, and / or components are present, but does not exclude the presence or addition of one or more other features, steps, operations, or components.

[0032] In agricultural production, rainwater erosion in the rainy season can seriously reduce the utilization rate of fungicides, and the existing technology is difficult to balance the persistence of drug efficacy and biological safety. In the process of realizing the present disclosure, it is found that a large number of network microstructures inside the liquid-liquid condensed phase have high encapsulation capacity for different types of drugs, and have certain viscosity and fluidity. However, the existing liquid-liquid condensed phase technology needs to use a large amount of synthetic surfactant, which leads to high biological toxicity and is difficult to promote and apply in the field of agriculture.

[0033] Based on this, the present disclosure constructs a liquid-liquid condensed phase by using a biological surfactant deoxycholic acid sodium and polyethyleneimine, and forms a rain-resistant fungicide system suitable for application in the rainy season. The selected biological surfactant has lower biological toxicity, and the formed liquid-liquid condensed phase is a full water-based system, which effectively avoids the biological safety hidden danger brought by traditional synthetic surfactants, and meets the development needs of green agriculture. And the preparation process is simple, without complex equipment and cumbersome steps, which is conducive to reducing production cost and suitable for large-scale production and application. The prepared liquid-liquid condensed phase can efficiently encapsulate drug molecules with different hydrophilic and hydrophobic properties, and can synergize with other pesticides to enhance the efficacy. At the same time, due to its high flexibility and rich network microstructure inside, it can match and entangle with the micro-nano structure of the hydrophobic leaf surface, significantly improving the rainwater resistance. In addition, the deoxycholic acid sodium / polyethyleneimine liquid-liquid condensed phase has broad-spectrum antibacterial activity, and when applied to the leaf surface of crops, it can effectively protect against common pathogenic bacteria even after rain erosion, fully meeting the application needs of rain-resistant fungicides in the rainy season.

[0034] According to an embodiment of one aspect of the present disclosure, a liquid-liquid condensed phase is provided, comprising: polyethyleneimine, deoxycholic acid sodium, wherein the concentration of polyethyleneimine is 0.5 g / L to 5 g / L, and the molecular weight is 600 to 25000; the concentration of deoxycholic acid sodium is 0.5 mM to 50 mM.

[0035] According to the embodiments of the present disclosure, the polyethyleneimine concentration may, for example, be 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, etc. If the concentration is too low, a sufficient network microstructure may not be formed, affecting drug encapsulation and leaf surface adhesion; if the concentration is too high, the system viscosity will be abnormal, which is not conducive to application and contact with the leaf surface. The polyethyleneimine molecular weight may be 600, 1000, 1500, 1800, 5000, 10000, 20000, 250000, etc. A suitable chain length and charge density of polyethyleneimine can fully interact with sodium deoxycholate to form a stable and flexible condensed phase. If the molecular weight is too small, the network structure is weak, and the rainwater resistance is reduced; if the molecular weight is too large, the system rigidity is enhanced, which is not conducive to matching and entanglement with the leaf micro-nano structure. The sodium deoxycholate concentration may, for example, be 0.5 mM, 1 mM, 5 mM, 10 mM, 30 mM, 50 mM, etc. An appropriate concentration can ensure its biological activity and, together with polyethyleneimine, ensure the broad-spectrum antibacterial ability of the liquid-liquid condensed phase.

[0036] According to the embodiments of the present disclosure, the present disclosure utilizes sodium deoxycholate and polyethyleneimine to construct a full water-based liquid-liquid condensed phase system. The polyethyleneimine molecular chain has a large number of positively charged amino groups, and the sodium deoxycholate molecule has a negatively charged sodium carboxylate group. Through various synergistic effects such as electrostatic interaction and hydrophobic interaction, a stable liquid-liquid condensed phase can be spontaneously formed. Sodium deoxycholate and polyethyleneimine both have low biological toxicity, effectively avoiding the potential biological toxicity problems brought by traditional organic silicon surfactants, and significantly improving environmental friendliness and biological safety.

[0037] Polyethyleneimine is a polymer with a multi-branched structure, and the sodium deoxycholate molecule structure simultaneously contains a hydrophobic steroid skeleton and a hydrophilic sodium carboxylate group. In the liquid-liquid condensed phase, the long-chain molecules of polyethyleneimine act as the skeleton of the network structure, and the sodium deoxycholate molecules are filled between the skeleton through hydrophobic and electrostatic interactions to form a large number of interconnected small gaps, thereby constructing a complex and stable network microstructure. Not only does this give the liquid-liquid condensed phase good encapsulation performance, but it also significantly improves the flexibility and wet surface adhesion of the liquid-liquid condensed phase. In addition, the polyethyleneimine molecular chain itself has a certain flexibility and can rotate and bend within a certain range. The interaction between the sodium deoxycholate molecules and the polyethyleneimine molecular chain further enhances the flexibility of the overall structure, allowing the liquid-liquid condensed phase to deform appropriately when subjected to external impact or water flow, thereby exhibiting good elasticity and plasticity, and thus being able to adapt to different micro-nano structure surfaces and effectively improve their rainwater resistance.

[0038] Further, as a bile acid derivative, the molecular structure of sodium deoxycholate can destroy the integrity of the fungal cell membrane, change the permeability of the cell membrane, and cause the leakage of internal substances of the fungus, thereby inhibiting the growth and reproduction of the fungus. At the same time, a large number of amino groups on the polyethyleneimine molecular chain have cationic antibacterial properties, which can interact with the anion components on the surface of the fungal cell membrane, interfere with the normal metabolic process of the fungus. The synergistic effect of the two makes the liquid-liquid coacervate itself have broad-spectrum bacteriostatic activity, and can effectively inhibit and kill a variety of common plant pathogenic fungi without additional addition of bactericides.

[0039] According to the embodiments of the present disclosure, the liquid-liquid coacervate further comprises a metal chloride salt. The metal chloride salt can shield the electrostatic interaction between the polyethyleneimine and the sodium deoxycholate molecules, increase the attractive force between the liquid-liquid coacervate droplets, further promote the coalescence of the coacervate droplets, and make the overall structure of the liquid-liquid coacervate more compact and uniform. At the same time, the addition of the metal chloride salt can also adjust the ionic strength of the system, change the solvation environment of the molecular chain, enhance the flexibility of the polyethyleneimine molecular chain, and further improve the overall flexibility of the liquid-liquid coacervate, so that the rainwater impact resistance of the liquid-liquid coacervate is further enhanced.

[0040] According to the embodiments of the present disclosure, the metal chloride salt comprises at least one of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and zinc chloride, and the concentration of the metal chloride salt is 0 mM to 500 mM, for example, 0 mM, 1 mM, 5 mM, 10 mM, 50 mM, 100 mM, 500 mM, etc. The metal chloride salt with an appropriate concentration can regulate the coalescence degree and flexibility of the liquid-liquid coacervate, optimize the comprehensive performance of the liquid-liquid coacervate, and make it more suitable for different application requirements in terms of rainwater impact resistance, drug encapsulation and release, etc.

[0041] According to the embodiments of the present disclosure, the diameter of the liquid-liquid coacervate is 300 nm to 30 μm, for example, 300 nm, 500 nm, 1 μm, 5 μm, 10 μm, 30 μm, etc. The liquid-liquid coacervate has a small size structure, which can adapt to the micro-nano structure characteristics of different crop leaf surfaces, can penetrate into the small gaps of the leaf surface, and form a larger contact area with the leaf surface, thereby enhancing the adhesion ability of the liquid-liquid coacervate.

[0042] According to the embodiment of the second aspect of the present disclosure, a preparation method of the liquid-liquid coacervate is provided, including: dissolving polyethyleneimine in water and adjusting pH to 6-8 to obtain a polyethyleneimine stock solution; mixing the polyethyleneimine stock solution with sodium deoxycholate and a metal chloride salt according to the corresponding concentration ranges, the polyethyleneimine concentration being 0.5 g / L-5 g / L, the molecular weight being 600-25000; the sodium deoxycholate concentration being 0.5 mM-50 mM; and the metal chloride salt concentration being 0 mM-500 mM, to obtain the liquid-liquid coacervate after stirring.

[0043] According to the embodiment of the present disclosure, the pH is adjusted to the near-neutral range of 6-8, the reaction condition is mild and easy to control, the destruction of the performance of raw materials by extreme conditions such as high temperature, high pressure or strong acid and alkali is avoided, and the formed liquid-liquid coacervate has a stable network microstructure. On this basis, the liquid-liquid coacervate can be prepared by mixing and stirring, the reaction process does not require complex reaction equipment and cumbersome operation steps, and the production difficulty and cost are greatly reduced.

[0044] According to the embodiment of the present disclosure, the mixing temperature is 15°C-30°C, the stirring speed is 500 rpm-2000 rpm, and the stirring time is 1 min-120 min. The reaction condition is simple, mild and easy to control, which is beneficial to industrial large-scale production.

[0045] To explore the preparation process of the liquid-liquid coacervate, different molecular weight polyethyleneimine (PEI600, PEI1800 and PEI10000) with concentrations of 1 g / L, 2 g / L, 3 g / L and 4 g / L were mixed with 100 mM sodium chloride (NaCl) solution in a double-layer glass protective sleeve. Then, a high-concentration sodium deoxycholate (NaDC) solution and an equal volume of 200 mM NaCl solution were added dropwise to the above-mentioned mixed solution, so that the concentration of NaDC in the system gradually increased, and the concentration of NaCl remained constant. The turbidity of the mixed solution was measured at a wavelength of 670 nm using a Brinkman PC920 probe colorimeter, and the initial turbidity value of the solution was set to zero. Figure 1 Turbidity curves of polyethyleneimine and sodium chloride mixed solutions with different molecular weights with increasing concentrations of sodium deoxycholate. Wherein a is the turbidity curve of the PEI600 and sodium chloride mixed solution with increasing concentrations of sodium deoxycholate; b is the turbidity curve of the PEI1800 and sodium chloride mixed solution with increasing concentrations of sodium deoxycholate; c is the turbidity curve of the PEI10000 and sodium chloride mixed solution with increasing concentrations of sodium deoxycholate.

[0046] As Figure 1As shown, with the increase of the concentration of sodium deoxycholate, the turbidity of the NaDC / PEI / NaCl mixed solution first experienced a slow and rapid increase, and remained basically unchanged after reaching the maximum. Correspondingly, from the change of turbidity and the corresponding interval of the micro-morphology characteristics, it can be known that the NaDC / PEI / NaCl mixed solution experienced four phase states of single-phase solution, complex, liquid-liquid condensed phase and precipitation. At low NaDC concentration, the turbidity of the system is close to the initial value, at this time, PEI and NaDC molecules are uniformly dispersed, and no obvious aggregation structure is formed; with the increase of the concentration of NaDC, the turbidity slowly rises, indicating that PEI and NaDC begin to form soluble complexes through electrostatic and hydrophobic interactions; when the concentration of NaDC reaches the critical value, the turbidity rapidly increases to the peak value, and a large number of stable liquid-liquid condensed phases are formed in the system, at this time, the PEI molecular chain as the skeleton is interwoven with NaDC molecules to form a network microstructure; when the concentration of NaDC is too high, the turbidity remains stable but the system appears macroscopic precipitation, because the excess NaDC destroys the balance of the condensed phase, leading to the instability of the structure and aggregation.

[0047] According to an embodiment of the third aspect of the present disclosure, a rain-resistant bactericide is provided, comprising the liquid-liquid condensed phase or the liquid-liquid condensed phase obtained by the preparation method.

[0048] According to an embodiment of the present disclosure, the rain-resistant bactericide of the present disclosure utilizes the rich network microstructure and high flexibility inside the liquid-liquid condensed phase, enabling it to closely match and entangle with the micro-nano structure of the hydrophobic crop leaf surface, thereby significantly improving the rainwater resistance of the bactericide on the leaf surface. Even in rainy or heavy rain weather conditions, the rain-resistant bactericide can effectively reduce the rebound and loss of liquid droplets, significantly improve the retention rate of the bactericide on the leaf surface, and ensure the long-term effectiveness and stability of the bactericide effect. In addition, the liquid-liquid condensed phase itself has broad-spectrum bacteriostatic activity, enabling the rain-resistant bactericide to effectively inhibit and kill a variety of common plant pathogens, and providing reliable disease protection for crops after being applied to the leaf surface of crops. At the same time, the rain-resistant bactericide is constructed using a low-biotoxicity biosurfactant, forming a full water-based system, and showing good safety and compatibility to crops, soil and ecological environment, meeting the needs of sustainable development of green agriculture.

[0049] According to an embodiment of the present disclosure, the rain-resistant bactericide also encapsulates hydrophilic drug molecules and / or hydrophobic drug molecules. The rain-resistant bactericide of the present disclosure comprises the aforementioned liquid-liquid condensed phase or the liquid-liquid condensed phase obtained by the aforementioned preparation method, and the polyethyleneimine is used as the skeleton in the liquid-liquid condensed phase, and the sodium deoxycholate molecules are filled between the skeletons to form a large number of interconnected small gaps, constituting a complex and stable network microstructure. Among them, the sodium deoxycholate molecule structure contains both hydrophobic steroids and hydrophilic sodium carboxylate groups, enabling the liquid-liquid condensed phase to efficiently encapsulate drug molecules with different hydrophilic and hydrophobic properties.

[0050] For hydrophilic drug molecules, they can be adsorbed in the network gap by interacting with the hydrophilic groups in the network structure; for hydrophobic drug molecules, they can be embedded in the region composed of hydrophobic groups by means of hydrophobic interaction, together with the liquid-liquid condensed phase with broad-spectrum antibacterial activity, to further enhance the killing range and inhibition effect on plant pathogenic bacteria.

[0051] According to the fourth aspect of the present disclosure, the application provides a use of the rain-resistant bactericide in agricultural sterilization.

[0052] According to the embodiments of the present disclosure, the rain-resistant bactericide of the present disclosure is suitable for disease control of various crops, and can meet the needs of pesticide application in rainy season or rainy areas. In the application process, the rain-resistant performance of the liquid-liquid condensed phase can reduce the loss of active ingredients under extreme weather such as heavy rain, ensure the retention rate on the leaf surface, and solve the problem of sudden decrease of drug efficacy caused by rain erosion of traditional bactericides, which requires frequent reapplication. The application frequency and drug dosage are significantly reduced, thereby saving the cost of agricultural production.

[0053] According to the embodiments of the present disclosure, the rain-resistant bactericide is used for inhibiting fungi on the leaves of crops, and the crops include at least one of rice, wheat, strawberry, pepper, cucumber, tomato, and apple, and the fungi include at least one of Rhizoctonia solani, Fusarium reticulatum, Fusarium graminearum, Magnaporthe grisea, Pyricularia grisea, Sclerotinia sclerotiorum, Phytophthora capsici, Valsa mali, Botrytis cinerea, Colletotrichum gloeosporioides, and Alternaria solani. The rain-resistant bactericide of the present disclosure can effectively deal with various common fungal diseases on crops and their leaves, and has significant broad-spectrum applicability. In actual application, it is not necessary to separately prepare the bactericide for different crops or pathogenic bacteria, which significantly reduces the difficulty of selection and use of the bactericide in agricultural production, and improves the convenience and efficiency of disease control.

[0054] In order to make the purpose, technical solutions and advantages of the present disclosure clearer, the present disclosure will be further described in detail below in combination with specific embodiments. If the specific technology or condition is not specified in the embodiments, it is a conventional method, which can be carried out according to the technology or condition described in the literature in the art or according to the product instruction.

[0055] Embodiment 1:

[0056] The present disclosure provides a liquid-liquid condensed phase, which is prepared by the following steps:

[0057] Polyethyleneimine (PEI) with a molecular weight of 600 is dissolved in water and the pH is adjusted to 7.5 to obtain a polyethyleneimine stock solution;

[0058] 2 g / L of polyethyleneimine mother liquor was mixed with 10 mM sodium deoxycholate (NaDC) and 100 mM sodium chloride (NaCl), and stirred at 500 rpm for 30 minutes to obtain liquid-liquid condensed phase 1 (NaDC / PEI600 / NaCl).

[0059] The morphology of liquid-liquid condensed phase 1 was observed using an optical microscope. Figure 2 This is a microscope image of the liquid-liquid condensed phase in Embodiment 1 of this disclosure.

[0060] like Figure 2 As shown, the liquid-liquid condensed phase exists in the bulk phase in the form of droplets with diameters ranging from several hundred nanometers to tens of micrometers.

[0061] Rhodamine 6G, calcein, and Nile Red fluorescent dye were added to the dispersion of liquid-liquid condensed phase 1 and shaken to obtain liquid-liquid condensed phase 1a encapsulated with Rhodamine 6G, liquid-liquid condensed phase 1b encapsulated with calcein, and liquid-liquid condensed phase 1c encapsulated with Nile Red.

[0062] Liquid-liquid condensed phases 1a-1c were each added in 20 μL to a glass slide and imaged using a laser confocal scanning microscope. The excitation wavelengths for Rhodamine 6G and Nile Red were λ. ex =559 nm, the excitation wavelength of calcein is λ ex =488 nm.

[0063] Figure 3 This is a confocal fluorescence image of the liquid-liquid condensed phase encapsulated with different fluorescent dyes in Example 1 of this disclosure.

[0064] like Figure 3 As shown, the liquid-liquid condensed phase droplets disclosed herein can encapsulate and enrich dyes with different hydrophilic and hydrophobic properties, such as the hydrophilic cationic dye Rhodamine 6G, the anionic dye Calcein, and the hydrophobic dye Nile Red. Because the liquid-liquid condensed phase simultaneously possesses a hydrophilic interface and hydrophobic microregions, and is not limited by a membrane structure, it can provide suitable binding sites and spaces for guest molecules with different charges and hydrophilic / hydrophobic properties, thereby efficiently enabling the entry and anchorage of various guest molecules and significantly improving the encapsulation compatibility and enrichment capability for different types of molecules.

[0065] Example 2:

[0066] This disclosure provides a liquid-liquid condensed phase, which differs from Example 1 in that: a liquid-liquid condensed phase 2 (NaDC / PEI1800 / NaCl) is prepared using polyethyleneimine with a molecular weight of 1800.

[0067] The morphology of liquid-liquid condensed phase 2 was observed using cryo-scanning electron microscopy. Figure 4 This is a scanning electron microscope image of the liquid-liquid condensed phase in Embodiment 2 of this disclosure.

[0068] As shown in Figure 4 , the liquid droplets of the liquid-liquid condensed phase coalesce to form micron-sized large droplets, and the condensed phase contains a large amount of network structure.

[0069] Example 3

[0070] The embodiment of the present disclosure provides a liquid-liquid condensed phase, which is different from the embodiment 1 in that polyethyleneimine with a molecular weight of 10000 is used, and a liquid-liquid condensed phase 3 (NaDC / PEI10000 / NaCl) is prepared.

[0071] Example 4

[0072] The embodiment of the present disclosure provides a liquid-liquid condensed phase, which is different from the embodiment 1 in that polyethyleneimine with a molecular weight of 1800 is used, and sodium chloride is not added during the preparation process, and a liquid-liquid condensed phase 4 (NaDC / PEI1800) is prepared.

[0073] Example 5

[0074] The embodiment of the present disclosure provides a liquid-liquid condensed phase, which is different from the embodiment 1 in that polyethyleneimine with a molecular weight of 10000 is used, and sodium chloride is not added during the preparation process, and a liquid-liquid condensed phase 5 (NaDC / PEI10000) is prepared.

[0075] The liquid-liquid condensed phase of the present disclosure is subjected to rainwater flushing test on different leaves.

[0076] 120 μL of the liquid-liquid condensed phase 2 (NaDC / PEI1800 / NaCl), the liquid-liquid condensed phase 3 (NaDC / PEI10000 / NaCl), and commercial adjuvants (Essential Oil, organic silicon adjuvant polyether modified heptamethyltrisiloxane (PMH), polyethyleneimine 1800 (PEI1800), and polyethyleneimine 10000 (PEI10000)) encapsulating 50 μM rhodamine 6G are respectively dropped on the surfaces of lotus leaves, rice leaves, wheat leaves, and cucumber leaves.

[0077] Figure 5a A schematic diagram of a rainwater flushing system.

[0078] As shown in FIG. 5, after the leaves are naturally dried, they are placed on an inclined plane with an inclination angle of 30°, and a rainwater flushing system shown in Figure 5a is used for flushing treatment, wherein the flushing rainfall is 400 mm·h -1The rinsing height was 30 cm. After rinsing for 2, 10, 30, 90 and 150 minutes, a 2 cm × 2 cm area covered with a fluorescent mark was cut from the leaf and placed in a live imaging system. Fluorescence imaging was performed under the conditions of excitation wavelength 570 nm and emission wavelength 620 nm, and the fluorescence radiation intensity was counted.

[0079] Figure 5b The images show the fluorescence of the liquid-liquid condensed phase and the commercially available adjuvant Rhodamine 6G on the surface of lotus leaves after being subjected to simulated rain. (Examples 2-3 of this disclosure)

[0080] As shown in Figure 5b, after Rhodamine 6G was coated onto liquid-liquid condensed phases 2 and 3 of this disclosure, the fluorescence radiation intensity after 150 minutes of rinsing was approximately 4.1 times and 3.5 times that of commercial adjuvants, respectively, indicating a significant reduction in fluorescent dye loss. Specifically, the fluorescence residue rates of commercial adjuvants PMH and plant essential oils decreased to 42.9% and 54.9% after 10 minutes of rinsing, and only 24.9% and 22.9% remained after 150 minutes. In contrast, liquid-liquid condensed phases 2 and 3 maintained residue rates of 66.9% and 68.4% respectively after rinsing on lotus leaves for 90 minutes, and their residue rates after 150 minutes were 12.3% and 10.3% higher than PMH, respectively. This demonstrates that the liquid-liquid condensed phases prepared with sodium deoxycholate and polyethyleneimine in this disclosure can reduce rainwater runoff, prolong the retention time on superhydrophobic leaf surfaces, and exhibit superior adhesion performance compared to commercial adjuvants.

[0081] Figure 5c The images show the fluorescence patterns of rice, wheat, and cucumber leaves after being subjected to simulated rain for different durations following the encapsulation of Rhodamine 6G in the liquid-liquid condensed phase and plant essential oil adjuvant in Examples 2-3 of this disclosure.

[0082] like Figure 5c As shown, the plant essential oil adjuvant loaded with Rhodamine 6G left only a small amount of fluorescent residue on the leaves of the three types of plants after 90 minutes of rinsing. In contrast, liquid-liquid condensed phases 2 and 3 loaded with Rhodamine 6G exhibited excellent rinsing resistance on the leaves of both rice and wheat, with significant fluorescent residue remaining even after 150 minutes of rinsing. The fluorescence radiation intensity on the rice leaves was approximately 1.1 × 10⁻⁶. 10 and 8.0×10 9 The wheat leaf surface area is approximately 3.6 × 10⁻⁶. 9 and 4.4×10 9 On cucumber leaves with relatively low hydrophobicity, liquid-liquid condensation phases 2 and 3 showed relatively weak resistance to rain washout, and the fluorescence residue basically disappeared after 90 minutes of washing.

[0083] The deposition morphology of the liquid-liquid condensate phase on the leaf surface of this disclosure is characterized.

[0084] The liquid-liquid condensed phase 2 (NaDC / PEI1800 / NaCl) of Example 2 was dropped on the surfaces of lotus leaf, rice, wheat and cucumber leaves respectively, and then the morphology was observed by scanning electron microscopy after natural deposition and drying. Figure 6 The scanning electron microscopy images of the liquid-liquid condensed phase of Example 2 before and after spraying on the surfaces of lotus leaf, rice, wheat and cucumber leaves.

[0085] As shown in Figure 6 , it can be observed that the liquid-liquid condensed phase is easy to coalesce and form large condensed bodies with flexibility. During the deposition process, for the super-hydrophobic leaf surface of lotus leaf and rice with dense and complex nano-microstructure and micrometer-sized papilla array, the liquid-liquid condensed phase is easy to deform after contact, matches the shape of the papilla, and promotes its adhesion on the papilla microstructure; at the same time, the condensed phase can also embed into the nano-microstructure gap of the leaf surface, entangle and tightly bind with the leaf surface through the internal network structure, and finally form a dense condensed film on the leaf surface, further improving the adhesion and rainwater resistance. For the wheat leaf surface, the cuticle lacks micrometer-sized papilla, resulting in that the liquid-liquid condensed phase and the leaf surface cannot achieve morphology matching, and the nano-microstructure density decreases, so that the entanglement of the liquid-liquid condensed phase network structure and the leaf surface is weakened, and only part of the area can be pinned and combined with the leaf surface, so the adhesion decreases, and the rainwater resistance is not as good as that on the lotus leaf and rice leaf surfaces. The cucumber leaf surface is more hydrophobic, has no nano-microstructure, and only has micrometer-sized folds, so the condensed phase cannot entangle with the leaf surface through the network structure, but can only stay on the leaf surface in the form of a spherical droplet, and thus the adhesion and rainwater resistance are the worst.

[0086] The adhesion between the liquid-liquid condensed phase of the present disclosure and the leaf surface was characterized. The test objects included the liquid-liquid condensed phases 2-5 of Examples 2-5 of the present disclosure, PEI1800 solution, PEI10000 solution and water.

[0087] Figure 7 The schematic diagram of the adhesion test system is shown in

[0088] As shown in Figure 7 , the adhesion test system was used to take 10 μL of the liquid to be tested and directly suspend it on a platinum ring with a diameter of 2 mm; or the dispersion liquid of the liquid-liquid condensed phases 2-5 was centrifuged at a speed of 7000 rpm for 10 minutes, and then 0.01 g of completely condensed phase droplet was taken and suspended on the platinum ring. The test process was as follows: the platform on which the leaf was placed was moved upward at a speed of 0.2 mm·s -1 -1 to contact the droplet, and after the platinum ring was immersed in the droplet by 0.3 mm, the platform was moved downward to separate from the droplet, and the adhesion change during the whole process was monitored and recorded synchronously to characterize the adhesion between the liquid-liquid condensed phase and the leaf surface. The adhesion between the droplet and different leaf surfaces was measured by using the adhesion tester.

[0089] Figure 8 Figure for the adhesion test of the liquid-liquid coacervate and its complete coacervate and water in Example 2 of the present disclosure.

[0090] As shown in FIG. 5, the liquid-liquid coacervate droplet of Example 2 of the present disclosure deformed significantly when it detached from the super-hydrophobic leaf surface due to the enhanced adhesion between the droplet and the leaf surface. However, the complete coacervate droplet was difficult to detach from the leaf surface due to the more compact network structure inside the droplet, the matching effect of the droplet with the leaf surface, and the micro-nano entanglement effect, which further strengthened the adhesion between the droplet and the leaf surface. Figure 8

[0091] Figure for the adhesion change curve of the liquid-liquid coacervate, the polyethyleneimine solution, and water in Examples 2-5 of the present disclosure. Figure 9 As shown in FIG. 6, the maximum adhesion between the water and the polyethyleneimine (PEI) solution and the lotus leaf surface was small, only about 0.01 mN, and the platinum ring stretching displacement when the droplet detached was only 0.65-0.71 mm. The adhesion between the NaDC / PEI1800 and NaDC / PEI10000 liquid-liquid coacervate and the lotus leaf was significantly improved, reaching 0.04 mN and 0.02 mN, respectively, and the stretching displacement when the droplet detached was extended to 0.87 and 0.73 mm. The addition of NaCl could promote the coalescence of the liquid-liquid coacervate droplet and improve its flexibility, making it easier to produce deposition deformation with the leaf surface topography. The maximum adhesion between the NaDC / PEI1800 / NaCl liquid-liquid coacervate and the lotus leaf reached 0.07 mN, and the stretching displacement when the droplet detached reached 1.57 mm, which were 7 times and 2.4 times that of water, respectively. The adhesion and deformation of the droplet on the rice leaf surface were greater than those on the lotus leaf because the rice leaf surface had more abundant and compact micro-nano papillae and ridge structures, and the structure matching effect between the droplet and the leaf surface was stronger. The adhesion between the droplet and the cucumber leaf surface was large due to the strong hydrophilicity of the cucumber leaf surface, which caused the contact area to increase rapidly, but the adhesion was still less than the adhesion between the droplet and the platinum ring, and the adhesion was easily washed off by rainwater after water evaporation due to the lack of micro-nano structure entanglement support.

[0092] Figure 9 The anti-fungal activity of the liquid-liquid coacervate of the present disclosure was tested.

[0093] The anti-fungal activity of the liquid-liquid coacervate of the present disclosure was tested.

[0094] ​The test method of antibacterial activity and broad spectrum of the liquid-liquid condensed phase and each component of the present disclosure is as follows: melt sterile potato dextrose agar (PDA) medium is diluted with sodium deoxycholate (NaDC), polyethyleneimine (PEI1800, PEI10000), and NaDC / PEI1800 / NaCl and NaDC / PEI10000 / NaCl to different concentrations, and then poured into a culture dish with a diameter of 90 mm. After the medium is solidified, a 5 mm diameter bacterial cake is inoculated in the center, and each group of treatments is repeated three times. After the inoculated medium is cultured at 25°C in the dark for 5 days, the colony diameters of different treatments are measured by cross method, and the inhibition rate is calculated according to formula (1) to test the antibacterial effect on different plant pathogenic fungi.

[0095] Formula (1)

[0096] Wherein C d (mm), T d (mm) and F d (mm) represent the colony diameter without treatment, the colony diameter of the sample treatment group, and the initial bacterial cake diameter, respectively.

[0097] Figure 10 The inhibition rate test diagram of the liquid-liquid condensed phase, sodium deoxycholate, polyethyleneimine, silicone adjuvant hydroxyalkynyl-terminated dimethylpolysiloxane and polyether-modified heptamethyltrisiloxane of the present disclosure embodiments 2-3 on Pyricularia oryzae. Among them, a is the inhibition rate test diagram of sodium deoxycholate and polyethyleneimine on Pyricularia oryzae; b is the inhibition rate test diagram of the liquid-liquid condensed phase, hydroxyalkynyl-terminated dimethylpolysiloxane, silicone adjuvant polyether-modified heptamethyltrisiloxane of the present disclosure embodiments 2-3 on Pyricularia oryzae.

[0098] As Figure 10 shown, among the materials for preparing the liquid-liquid condensed phase, NaDC, PEI1800 and PEI10000 can significantly inhibit the mycelial growth and spread of Pyricularia oryzae, in addition to sodium chloride, wherein the inhibition rates of 10 mM NaDC, 2 g·L -1 of PEI1800 and PEI10000 are close to 100%. Although the antibacterial activity of each component decreases with decreasing concentration, NaDC still maintains an inhibition rate of 58.5% after being diluted by 20 times (about 0.2 g·L -1 ), and the inhibition rates of PEI1800 and PEI10000 after being diluted by 20 times (about 0.1 g·L -1 ) are 81.4% and 74.4% respectively, still maintaining high activity. The liquid-liquid condensed phase 2-3 of the present disclosure (mass concentration about 12 g·L -1) can completely inhibit the growth of rice flat nematode, and the antibacterial activity is better than that of organic silicon additives PMH and CTP at the same concentration; especially under low concentration conditions (diluted by 120 times to 0.1 g·L -1 ), the antibacterial rates of NaDC / PEI1800 / NaCl and NaDC / PEI10000 / NaCl systems are 43.6% and 42.0% respectively, which are significantly higher than those of PMH (33.2%) and CTP (4.5%).

[0099] Figure 11 The antibacterial rate diagram of the liquid-liquid condensed phase, sodium deoxycholate, polyethyleneimine and sodium chloride of embodiments 2-3 of the present disclosure on 11 common plant pathogenic fungi. Among them, a is the antibacterial rate diagram of the liquid-liquid condensed phase of embodiments 2-3 of the present disclosure on 11 common plant pathogenic fungi; b is the antibacterial rate diagram of sodium deoxycholate, polyethyleneimine and sodium chloride on 11 common plant pathogenic fungi.

[0100] As shown in Figure 11 , the diluted liquid-liquid condensed phase 2-3 system shows good antibacterial effect on a variety of plant pathogenic fungi, among which the antibacterial rates on Rhizoctonia solani, Fusarium oxysporum, Fusarium graminearum, Magnaporthe oryzae, Pyricularia allii-populineum, Sclerotinia sclerotiorum, Phytophthora capsici, Valsa mali and Botrytis cinerea are all above 74.0%; the antibacterial activity on Colletotrichum gloeosporioides and Alternaria solani is relatively weak, but the minimum is still 46.7%, which is related to the difference in inhibitory activity of NaDC, PEI1800 and PEI10000 on different fungi. The corresponding concentration of NaDC has an antibacterial rate of about 75.0% on five kinds of fungi such as Rhizoctonia solani, so the antibacterial rate of the liquid-liquid condensed phase 2-3 on them is basically 73.8-94.1%; the antibacterial rate of NaDC on four kinds of fungi such as Sclerotinia sclerotiorum is close to 90.0%, and the antibacterial rate of different molecular weight PEI is 47.0-98.1%, so the antibacterial activity of the liquid-liquid condensed phase 2-3 on these four fungi is the highest, reaching more than 87.0%; and because the antibacterial rates of NaDC and PEI on Colletotrichum gloeosporioides and Alternaria solani are only 5.0-64.2%, the antibacterial activity of the liquid-liquid condensed phase 2-3 on these two fungi is relatively low.

[0101] Rainwater flushing antibacterial test was conducted on the liquid-liquid condensed phase of the present disclosure.

[0102] The liquid-liquid condensed phase dispersions of embodiments 2 and 4 of the present disclosure were uniformly sprayed on rice seedlings. After natural air drying, the seedlings were respectively subjected to 400 mm·h -1The rainstorm of 30 minutes, 60 minutes and 90 minutes, and the rainstorm after inoculation of Rhizoctonia solani mycelium suspension, and the moisture culture until the plant disease and the investigation of the disease condition. The disease is divided into 10 levels, wherein, 0 level is no disease spot; 1 level is disease spot area accounts for 5% or less of the whole leaf area; 3 level is disease spot area accounts for 6~10% of the whole leaf area; 5 level is disease spot area accounts for 11~20% of the whole leaf area; 7 level is disease spot area accounts for 21~40% of the whole leaf area; 9 level is disease spot area accounts for 40% or more of the whole leaf area. The disease index and the disease prevention effect are calculated according to formula (2) and formula (3) respectively.

[0103] Formula (2)

[0104] Formula (3)

[0105] Figure 12 The liquid-liquid condensed phase of the present disclosure is tested for the antibacterial effect on Rhizoctonia solani after rainwater flushing on the surface of rice leaves in example 2 and example 4 of the present disclosure.

[0106] As shown in Figure 12 , the liquid-liquid condensed phase of the present disclosure has a significant prevention effect on rice sheath blight. The surface of the rice leaves sprayed with water only has multiple large disease spots, and the disease degree of the plants sprayed with the liquid-liquid condensed phase in example 2 and example 4 of the present disclosure is significantly reduced. The prevention effect of the rice after applying the liquid-liquid condensed phase without rainwater flushing can reach more than 95%, although the prevention effect will decrease with the increase of the rainwater flushing time, the liquid-liquid condensed phase 5 system of the present disclosure has strong rainwater resistance, and the influence of rainwater flushing on the prevention effect is low, and the system can still maintain a prevention effect of 55.7% after 90 minutes of rainwater flushing.

[0107] The liquid-liquid condensed phase of the present disclosure is tested for the toxicity to seed germination.

[0108] The liquid-liquid condensed phase of example 2~3 of the present disclosure, the organic silicon auxiliary agent PMH and the CTP solution are diluted to 100 mg·L -1 , respectively, and 10 mL of each is added to a culture dish with a diameter of 90 mm and covered with double-layer filter paper. After the wheat seeds are pre-soaked for 24 hours, 10 seeds are scattered into the culture dish, and the culture is carried out at 25°C for 3 days. The pure water treatment is used as a blank control, and each group of experiments is repeated 3 times. The average values of the germination rate, root length and sprout length are measured and calculated, and single factor variance analysis is carried out through analysis software, and LSD and Dunnett T3 test method (significance level is 0.05) is used, wherein different lowercase letters represent significant difference (P<0.05).

[0109] Figure 13The germination rate, root length, and shoot length of wheat seeds cultured in the liquid-liquid condensed phase of Examples 2-3 of this disclosure and the diluted solution of organosilicon additives polyether-modified heptamethyltrisiloxane and hydroxyynyl-terminated dimethylpolysiloxane are statistical graphs.

[0110] like Figure 13 As shown, although the liquid-liquid condensate phases of Examples 2-3 of this disclosure have a certain inhibitory effect on wheat germination, the degree of inhibition is significantly lower than that of the commercial organosilicon adjuvant PMH at the same concentration. Wheat seeds treated with the liquid-liquid condensate phase exhibited a germination rate of 83.3-86.7%, an average root length of 2.21-2.49 cm, and an average shoot length of 2.30-2.45 cm, showing a smaller decrease compared to the pure water control (100% germination rate, 3.98 cm root length, and 2.94 cm shoot length). In contrast, treatment of wheat seeds with the same concentration of commercial organosilicon resulted in a decrease in germination rate, root length, and shoot length to 16.7-33.3%, 0.12-0.90 cm, and 0.24-1.05 cm, respectively. Therefore, the liquid-liquid condensate phase of this disclosure exhibits significantly lower toxicity to wheat seeds than commonly used commercial organosilicon adjuvants, demonstrating superior biocompatibility.

[0111] The specific embodiments described above further illustrate the purpose, technical solutions, and beneficial effects of this disclosure. It should be understood that the above descriptions are merely specific embodiments of this disclosure and are not intended to limit this disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.

Claims

1. A liquid-liquid condensed phase, characterized in that, The liquid-liquid condensed phase comprises: polyethyleneimine and sodium deoxycholate, wherein... The polyethyleneimine concentration is 0.5 g / L to 5 g / L, and the molecular weight is 600 to 25000; The concentration of sodium deoxycholate is 0.5 mM to 50 mM.

2. The liquid-liquid condensed phase according to claim 1, characterized in that, The liquid-liquid condensed phase also includes metal chloride salts.

3. The liquid-liquid condensed phase according to claim 2, characterized in that, The metal chloride salt includes at least one of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and zinc chloride; The concentration of the metal chloride salt is 0 mM to 500 mM.

4. The liquid-liquid condensed phase according to claim 1, characterized in that, The diameter of the liquid-liquid condensed phase is 300 nm to 30 μm.

5. The method for preparing a liquid-liquid condensed phase according to any one of claims 1 to 4, characterized in that, The preparation method includes: Polyethyleneimine was dissolved in water and the pH was adjusted to 6-8 to obtain a polyethyleneimine mother liquor. The polyethyleneimine mother liquor was mixed with sodium deoxycholate and a metal chloride salt, and the mixture was stirred to obtain the liquid-liquid condensed phase.

6. The preparation method according to claim 5, characterized in that, The mixing temperature is 15℃~30℃. The stirring speed is 500 rpm to 2000 rpm, and the stirring time is 1 min to 120 min.

7. A rain-resistant bactericide, characterized in that, The rain-resistant bactericide includes the liquid-liquid condensate phase as described in any one of claims 1 to 4, or the liquid-liquid condensate phase obtained by the preparation method described in claim 5 or 6.

8. The rain-resistant bactericide according to claim 7, characterized in that, The rain-resistant bactericide also encapsulates hydrophilic and / or hydrophobic drug molecules.

9. The application of a rain-resistant bactericide as described in any one of claims 7 to 8 in agricultural sterilization.

10. The application according to claim 9, characterized in that, The rain-resistant fungicide is used to inhibit fungi on crop leaves. The crops include at least one of rice, wheat, strawberries, peppers, cucumbers, tomatoes, and apples. The fungi include at least one of Rhizoctonia solani, Fusarium oxysporum, Fusarium graminearum, rice blast fungus, Cynosporium scutellatus, Phytophthora capsici, Phytophthora macrantha, Botrytis cinerea, Anthracis pilosa, and Alternaria alternata.

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