Preparation method of plant extract compound tea tree oil bactericide

Abstract

This invention discloses a method for preparing a plant extract compound tea tree oil fungicide, relating to the field of agricultural chemical formulation technology. The method includes preparing extracts of Sophora flavescens, Scutellaria baicalensis, and Galla chinensis polyphenols, and determining the amounts of each at a solid-to-mass ratio of 1:(0.8–1.5):(0.3–0.8). This invention improves the coating stability of the tea tree oil and plant extract compound system by constructing a Galla chinensis polyphenol interface layer and a calcium-crosslinked pectin outer wall on the surface of tea tree oil-containing latex droplets. Simultaneously, this calcium-crosslinked pectin outer wall can promote the release of active ingredients in the presence of oxalic acid or citric acid and pectin-degrading enzymes, thereby improving the fungicide's inhibitory effect on Botrytis cinerea.

Description

Technical Field

[0001] This invention relates to the field of agricultural chemical formulation technology, and in particular to a method for preparing a fungicide by combining plant extracts with tea tree oil. Background Technology

[0002] Plant-derived fungicides, due to their natural origin, good environmental compatibility, and diverse target sites, have become a core direction in the research and development of green agricultural inputs. Encapsulating active substances with wall materials can achieve multiple objectives, including environmental isolation, controlled release, and improved stability.

[0003] Early research often used synthetic polymer wall materials, but in recent years there has been a greater shift towards widely available, biodegradable natural polymer materials, such as pectin, chitosan, and starch.

[0004] However, existing plant-derived fungicide microcapsules mainly focus on improving encapsulation efficiency and storage stability, and their release behavior usually only responds to a single environmental factor. For highly volatile and hydrophobic active ingredients such as tea tree oil, ordinary emulsification systems are prone to problems such as volatilization, leakage, or stratification. Conventional natural polymer wall materials are difficult to achieve effective release under the coexistence of organic acids and pectin-degrading enzymes while ensuring storage stability. Therefore, how to obtain a plant-derived compound fungicide with good stability and that can promote release under the above coexistence conditions is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: This invention provides a method for preparing a plant extract compound tea tree oil bactericide, comprising: S1. Prepare extracts of Sophora flavescens, Scutellaria baicalensis, and Galla chinensis polyphenols, and determine the dosage of the three extracts according to a solid mass ratio of 1:(0.8-1.5):(0.3-0.8). S2, using low-methoxylated citrus pectin and gallic acid as raw materials, gallic acid-grafted low-methoxylated citrus pectin is prepared, wherein the degree of esterification of the low-methoxylated citrus pectin is 20%–35%, and the grafting rate of the gallic acid-grafted low-methoxylated citrus pectin is 10%–25%; S3, tea tree oil, sophora flavescens extract, scutellaria baicalensis extract, tea saponin and soybean lecithin were added to an aqueous phase with a pH of 6.2 to 6.8 and emulsified to obtain a tea tree oil-plant extract emulsion; S4, add gallnut polyphenol extract to tea tree oil-plant extract emulsion, so that gallnut polyphenols are bound to the surface of emulsion droplets to form a polyphenol interface layer; S5, add gallic acid-grafted low-methoxy citrus pectin to the emulsion that forms a polyphenol interface layer, so that the gallic acid-grafted low-methoxy citrus pectin is deposited on the outside of the polyphenol interface layer. S6, add calcium chloride solution to crosslink calcium ions with carboxyl groups in gallic acid-grafted low-methoxy citrus pectin, forming a calcium crosslinked pectin outer wall on the outside of the droplets, to obtain a microcapsule-type plant extract compound tea tree oil bactericide. The microcapsule-type plant extract compound tea tree oil bactericide includes tea tree oil-containing emulsion droplets, a polyphenol interface layer, and a calcium cross-linked pectin outer wall, and contains Sophora flavescens extract and Scutellaria baicalensis extract. The polyphenol interface layer is bonded to the surface of the tea tree oil-containing emulsion droplets, and the calcium cross-linked pectin outer wall is located outside the polyphenol interface layer. The outer wall of the calcium-crosslinked pectin includes a crosslinked structure formed by grafting gallic acid onto carboxyl groups and calcium ions in low-methoxy citrus pectin. The crosslinked structure can be weakened in the presence of oxalic acid or citric acid, and the pectin backbone in the outer wall of the calcium-crosslinked pectin can be cleaved in the presence of pectin-degrading enzymes.

[0006] Preferably, in S1, the Sophora flavescens extract is obtained by extraction with a 60%–70% ethanol aqueous solution, the Scutellaria baicalensis extract is obtained by extraction with a 50%–70% ethanol aqueous solution, and the Galla chinensis polyphenol extract is obtained by extraction with a 30%–50% ethanol aqueous solution.

[0007] Preferably, in S1, the solid mass ratio of the Sophora flavescens extract, Scutellaria baicalensis extract and Galla chinensis polyphenol extract is 1:(1-1.2):(0.4-0.6).

[0008] Preferably, in S2, the molar ratio of gallic acid to the galacturonic acid unit in the low-methoxy citrus pectin is (0.10-0.30):1.

[0009] Preferably, in S2, the gallic acid-grafted low-methoxy citrus pectin is prepared by a laccase-catalyzed grafting reaction.

[0010] Preferably, in S3, the mass ratio of tea saponin to soybean lecithin is 1:(0.5-2).

[0011] Preferably, in S3, the mass ratio of the tea tree oil to the total solids of the sophora flavescens extract and scutellaria baicalensis extract is 10:(1-4).

[0012] Preferably, in step S4, the amount of gallnut polyphenol extract added is 3% to 10% of the mass of tea tree oil.

[0013] Preferably, in S5, the dry basis amount of gallic acid-grafted low-methoxy citrus pectin is 4% to 12% of the mass of tea tree oil.

[0014] Preferably, in S6, the molar ratio of calcium ions to carboxyl groups in the gallic acid-grafted low-methoxy citrus pectin is (0.20–0.50):1.

[0015] The beneficial effects of this invention are as follows: By constructing a gallnut polyphenol interface layer and a calcium-crosslinked pectin outer wall on the surface of tea tree oil-containing latex droplets, this invention helps to improve the coating stability and storage stability of the tea tree oil and plant extract compound system; at the same time, the calcium-crosslinked pectin outer wall can promote the release of active ingredients under the condition of oxalic acid or citric acid and pectin degrading enzymes, thereby improving the inhibitory effect of fungicides on gray mold. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a flowchart illustrating the preparation process of the plant extract compound tea tree oil bactericide in Example 1.

[0018] Figure 2 This is a schematic diagram of the microcapsule hierarchical structure in Example 1.

[0019] Figure 3 This is a diagram illustrating the release mechanism of oxalic acid or citric acid in Example 1, where oxalic acid or citric acid interacts with pectin-degrading enzymes. Detailed Implementation

[0020] To make the above-mentioned objectives, features and advantages of the present invention more readily understood, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0021] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0022] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0023] Example 1, referring to Figures 1 to 3This is the first embodiment of the present invention, which provides a method for preparing a plant extract compound tea tree oil bactericide, including the following steps: This embodiment provides a method for preparing a plant extract compound tea tree oil bactericide. The method includes preparing plant extracts, preparing gallic acid grafted with low-methoxy citrus pectin, emulsifying tea tree oil and plant extracts, forming a polyphenol interface layer, depositing grafted pectin, and forming a calcium cross-linked pectin outer wall.

[0024] In this embodiment, the solid mass of the plant extract is measured on a dry basis. 5.00g of plant extract is placed in a pre-weighed weighing dish and dried at 105°C until the difference between two consecutive weighings does not exceed 0.005g. The mass of the dried residue is then used as the solid mass.

[0025] In this embodiment, the degree of esterification of low-methoxy citrus pectin was determined by acid-base titration. During the determination, 0.500 g of dried pectin sample was weighed and dissolved in deionized water. First, the free carboxyl groups were titrated with 0.100 mol / L NaOH standard solution and the volume consumed was recorded as V1. Then, excess NaOH solution was added to saponify the esterified carboxyl groups. After neutralization with hydrochloric acid, the released carboxyl groups were titrated with 0.100 mol / L NaOH standard solution and the volume consumed was recorded as V2. The degree of esterification DE was calculated as DE = V2 / (V1+V2)×100%.

[0026] In this embodiment, the gallic acid grafting rate refers to the percentage of the molar amount of gallic acid grafted onto low-methoxylated citrus pectin relative to the molar amount of galacturonic acid units in the low-methoxylated citrus pectin.

[0027] In this embodiment, the gallic acid grafting rate was determined by the Folin-Ciocalteu method. During the determination, unreacted free gallic acid was first removed by washing with ethanol and dialysis. Then, a standard curve was established using gallic acid standards to determine the gallic acid content in the grafted product. The grafting rate was calculated based on the ratio of the molar amount of gallic acid in the grafted product to the molar amount of galacturonic acid units in low-methoxy citrus pectin.

[0028] In this embodiment, the molar amount of galacturonic acid units in low-methoxylated citrus pectin was calculated based on the dry basis mass of low-methoxylated citrus pectin and the galacturonic acid content. The galacturonic acid content can be determined by the carbazole sulfuric acid method.

[0029] In this embodiment, the molar ratio of calcium ions to carboxyl groups in gallic acid-grafted low-methoxy citrus pectin is calculated based on the molar amount of calcium ions provided by the amount of calcium chloride added and the molar amount of carboxyl groups corresponding to the unesterified galacturonic acid units in the grafted pectin.

[0030] In this embodiment, the tea tree oil release rate is characterized by the release amount of the characteristic component terpinen-4-ol in tea tree oil. The terpinen-4-ol content in the release medium is determined by gas chromatography, and the percentage of the mass of terpinen-4-ol in the release medium to the total mass of terpinen-4-ol in the sample is used as the tea tree oil release rate.

[0031] In this embodiment, oligogalacturonic acid fragments with a degree of polymerization of 6 to 12 were detected by high performance liquid chromatography, and the degree of polymerization range was determined based on the retention time of oligogalacturonic acid standards.

[0032] In this embodiment, the calcium ion cross-linking structure in the outer wall of the calcium cross-linked pectin refers to the cross-linking structure formed by calcium ions and gallic acid grafted onto the unesterified carboxyl groups in low-methoxy citrus pectin.

[0033] In this embodiment, the weakening of calcium ion cross-linking structure by oxalic acid or citric acid means that after oxalic acid or citric acid binds to calcium ions, it reduces the cross-linking maintenance effect of calcium ions on pectin carboxyl groups.

[0034] In this embodiment, the pectin backbone is used to be cleaved by pectin-degrading enzymes. This means that the galacturonic acid backbone in the outer wall of calcium-crosslinked pectin is broken under the action of pectin-degrading enzymes and generates oligogalacturonic acid fragments.

[0035] In this embodiment, the polyphenol interface layer is located between the tea tree oil-containing latex droplets and the outer wall of the calcium cross-linked pectin, which is used to improve the stability of the latex droplet interface and provide deposition sites for gallic acid-grafted low-methoxy citrus pectin.

[0036] The dried Sophora flavescens herb was pulverized and passed through a 60-mesh sieve. 65% ethanol aqueous solution was added, and the mixture was extracted at 60℃ for 2 hours at a material-to-liquid ratio of 1:10 g / mL. After filtration, the residue was extracted again under the same conditions. The filtrates were combined and concentrated under reduced pressure at 50℃ and then freeze-dried to obtain a Sophora flavescens extract with a solids yield of 18.7%.

[0037] The dried Scutellaria baicalensis was pulverized and passed through a 60-mesh sieve. 60% ethanol aqueous solution was added, and the mixture was extracted at 60℃ for 2 hours at a material-to-liquid ratio of 1:10 g / mL. After filtration, the residue was extracted again under the same conditions. The filtrates were combined and concentrated under reduced pressure at 50℃ and then freeze-dried to obtain a Scutellaria baicalensis extract with a solids yield of 23.4%.

[0038] Dried gallnuts were pulverized and passed through a 60-mesh sieve. 40% ethanol aqueous solution was added, and the mixture was extracted at 50℃ for 1.5h at a material-to-liquid ratio of 1:12 g / mL. After filtration, the residue was extracted again under the same conditions. The filtrates were combined, concentrated under reduced pressure at 45℃, and freeze-dried to obtain a gallnut polyphenol extract with a solids yield of 46.2% and a total polyphenol content of 61.8%.

[0039] Weigh out 1.19g of Sophora flavescens extract, 1.31g of Scutellaria baicalensis extract, and 0.60g of Galla chinensis polyphenol extract according to the solid mass ratio, so that the solid mass ratio of Sophora flavescens extract, Scutellaria baicalensis extract and Galla chinensis polyphenol extract is 1:1.1:0.5.

[0040] In this embodiment, Sophora flavescens extract and Scutellaria baicalensis extract are used to form a compound bactericidal active system with tea tree oil, and Galla chinensis polyphenol extract is used to form a polyphenol interface layer on the surface of the droplets for grafting pectin deposition.

[0041] Take low-methoxylated citrus pectin with a degree of esterification of 28.3%, add it to sodium acetate buffer solution at pH 5.5, and prepare a pectin solution with a mass fraction of 1.5%.

[0042] Gallic acid was added to the pectin solution to make the molar ratio of gallic acid to galacturonic acid units in low-methoxy citrus pectin 0.20:1. Then, laccase with a dosage of 80 U / g pectin was added, and the reaction was carried out at 35℃ and 300 r / min for 6 h.

[0043] After the reaction was completed, the reaction solution was added to 3 times the volume of anhydrous ethanol for alcohol precipitation. After centrifugation, the precipitate was collected and washed 3 times with 60% ethanol aqueous solution. The precipitate was then redissolved in deionized water and dialyzed for 24 hours. After freeze drying, gallic acid-grafted low-methoxy citrus pectin was obtained.

[0044] The grafting rate of gallic acid grafted with low-methoxy citrus pectin was determined using the Folin-Ciocalteu method, and the grafting rate was found to be 17.8%.

[0045] In this embodiment, the degree of esterification of low-methoxy citrus pectin is used to ensure that the pectin has carboxyl segments that can crosslink with calcium ions, and the gallic acid grafting rate is used to ensure that the grafted pectin can be deposited on the outside of the gallnut polyphenol interface layer and retain the carboxyl sites required for calcium crosslinking.

[0046] Take 10.0g of tea tree oil, 1.19g of sophora flavescens extract, 1.31g of scutellaria baicalensis extract, 0.35g of tea saponin and 0.35g of soybean lecithin, add the above materials to 80.0g of deionized aqueous phase at pH 6.5, and shear at high speed for 10min at 8000r / min to obtain tea tree oil-plant extract emulsion.

[0047] In this embodiment, the mass ratio of tea tree oil to the total solids of Sophora flavescens extract and Scutellaria baicalensis extract is 10:2.5, and the mass ratio of tea saponin to soybean lecithin is 1:1.

[0048] 0.6g of gallnut polyphenol extract was dissolved in 5.40g of deionized water to prepare a 10.0% (w / w) gallnut polyphenol extract aqueous solution. This aqueous solution was then added dropwise to a tea tree oil-plant extract emulsion. During the dropwise addition, the stirring speed was controlled at 500r / min and the dropwise addition time was 20min. After the dropwise addition was completed, stirring was continued for 30min to allow the gallnut polyphenols to bind to the surface of the emulsion droplets and form a polyphenol interface layer.

[0049] The polyphenol interface layer is used to improve the interfacial stability of tea tree oil-plant extract droplets and to provide binding sites for the deposition of gallic acid-grafted low-methoxy citrus pectin on the outer side of the droplets.

[0050] 0.6 g of gallic acid-grafted low-methoxylated citrus pectin was dissolved in 59.40 g of deionized water to prepare a 1.0% (w / w) grafted pectin aqueous solution. This solution was then added dropwise to the emulsion forming a polyphenol interface layer under stirring at 400 r / min for 20 min. After the addition was complete, stirring was continued for 40 min to allow the gallic acid-grafted low-methoxylated citrus pectin to deposit on the outer side of the polyphenol interface layer.

[0051] Gallic acid-grafted low-methoxylated citrus pectin is deposited on the outer side of the polyphenol interface layer. This allows the cross-linking reaction to mainly occur on the outer side of the droplets and form the pectin outer wall during the subsequent calcium ion cross-linking process, thereby improving the stability of the tea tree oil and plant extract compound system.

[0052] In this embodiment, the galacturonic acid content of the low-methoxy citrus pectin was measured to be 80.0%. Based on the molar mass of anhydrous galacturonic acid unit of 176.12 g / mol, the molar amount of galacturonic acid unit in 0.6 g gallic acid-grafted low-methoxy citrus pectin is 2.73 mmol. Combined with the degree of esterification of 28.3%, the molar amount of carboxyl groups that can participate in calcium crosslinking is calculated to be 1.95 mmol.

[0053] Based on a calcium ion to carboxyl group molar ratio of 0.35:1, the required amount of calcium ions is 0.68 mmol.

[0054] Add 6.8 mL of 0.1 mol / L calcium chloride solution to the above system dropwise over a period of 20 min. After the addition is complete, continue stirring for 30 min to allow calcium ions to crosslink with the carboxyl groups of pectin and form a calcium-crosslinked pectin outer wall on the outside of the droplets.

[0055] After cross-linking, the resulting system was filtered through a 100-mesh sieve to obtain a microcapsule-type plant extract compound tea tree oil bactericide.

[0056] The resulting microcapsule-type plant extract compound tea tree oil bactericide includes tea tree oil-containing emulsion droplets, a polyphenol interface layer, and a calcium-crosslinked pectin outer wall, and also contains Sophora flavescens extract and Scutellaria baicalensis extract.

[0057] The bactericide obtained in this embodiment was tested and found to have an average particle size of 2.36 μm, a PDI of 0.214, and a tea tree oil encapsulation rate of 86.7%. It showed no obvious stratification after centrifugation at 3000 r / min for 30 min and no obvious demulsification after heat storage at 54℃ for 14 days.

[0058] The bactericide obtained in this embodiment was placed in a pH 6.5 buffer solution for 24 hours, and the release amount was measured to be 11.8% using terpinen-4-ol from tea tree oil as the characteristic component.

[0059] The bactericide obtained in this embodiment was placed in a pH 4.8 hydrochloric acid buffer and treated with pectin-degrading enzyme for 24 hours. The release of characteristic components of tea tree oil was measured to be 29.4%.

[0060] The bactericide obtained in this embodiment was placed in a pH 4.8 oxalate buffer solution and treated with pectin-degrading enzyme for 24 hours. The release of characteristic components of tea tree oil was measured to be 81.5%.

[0061] Under the same pH conditions, the amount of tea tree oil released when treated with oxalic acid and pectin-degrading enzymes was higher than that when treated with hydrochloric acid and pectin-degrading enzymes, indicating that the release of the calcium-crosslinked pectin outer wall obtained in this example is related to the weakening of the calcium crosslinking structure by oxalic acid.

[0062] High-performance liquid chromatography (HPLC) was used to detect oligogalacturonic acid fragments with a degree of polymerization of 6 to 12 in the release solution after co-treatment with oxalic acid and pectin degrading enzyme.

[0063] Using gray mold as the test object, and converting different formulations to the same effective addition amount according to the tea tree oil content, the fungicide obtained in this example showed an inhibition rate of 86.2% on the mycelial growth of gray mold and an inhibition rate of 88.7% on the spore germination of gray mold.

[0064] The aforementioned gray mold fungus is also known as Botrytis cinerea, and its treasure code is CGMCC3.3789.

[0065] In a simulated experiment of gray mold disease on detached tomato leaves, the fungicide obtained in this embodiment was sprayed onto the leaf surface and then inoculated with a suspension of gray mold spores. The leaves were cultured at 25°C and relative humidity above 90% for 72 hours, and the diameter of the lesions was measured to be 6.9 mm.

[0066] The results of this embodiment show that by combining tea tree oil with Sophora flavescens extract and Scutellaria baicalensis extract, and constructing a polyphenol interface layer and a calcium-crosslinked pectin outer wall, the resulting bactericide has storage stability, release capacity under the combined action of oxalic acid and pectin degrading enzymes, and inhibitory effect on gray mold.

[0067] Example 2 This embodiment illustrates that, even when the amount of grafted pectin added and the ratio of calcium ion crosslinking are close to the lower limit, it is still possible to prepare a plant extract compound tea tree oil bactericide with a calcium crosslinked pectin outer wall and responsive release ability.

[0068] Except for the parameter adjustments described below, all other extraction, concentration, drying, grafting, emulsification, interface layer formation, pectin deposition, calcium crosslinking, filtration, and detection methods in this embodiment are performed in accordance with Example 1.

[0069] In the preparation of plant extracts, Sophora flavescens extract was obtained by extraction with 60% ethanol aqueous solution, Scutellaria baicalensis extract was obtained by extraction with 50% ethanol aqueous solution, and Galla chinensis polyphenol extract was obtained by extraction with 30% ethanol aqueous solution.

[0070] Based on 10.0g of tea tree oil, 0.66g of Sophora flavescens extract, 0.54g of Scutellaria baicalensis extract and 0.34g of Galla chinensis polyphenol extract were weighed out, so that the mass ratio of tea tree oil to the total solids of Sophora flavescens extract and Scutellaria baicalensis extract was 10:1.2, and the amount of Galla chinensis polyphenol extract added was 3.4% of the mass of tea tree oil.

[0071] In this embodiment, the solid mass ratio of Sophora flavescens extract, Scutellaria baicalensis extract and Galla chinensis polyphenol extract is 1:0.82:0.52.

[0072] In this embodiment, the amount of tea saponin used is 0.35g, the amount of soybean lecithin used is 0.21g, and the mass ratio of tea saponin to soybean lecithin is 1:0.6.

[0073] In this embodiment, the amount of gallnut polyphenol extract used is close to the lower limit of the scope defined by the present invention, which is used to verify that an interface basis for grafting pectin deposition can still be formed on the surface of the emulsion droplets even with a lower amount of polyphenol interface material.

[0074] In the preparation of gallic acid-grafted low-methoxylated citrus pectin, low-methoxylated citrus pectin with an esterification degree of 21.4% was selected, and laccase-catalyzed grafting reaction was carried out according to a molar ratio of gallic acid to galacturonic acid units of 0.10:1.

[0075] The grafting rate of gallic acid-grafted low-methoxy citrus pectin was 11.6% as determined by the Folin-Ciocalteu method.

[0076] In this embodiment, a lower degree of esterification is used to provide more carboxyl crosslinking sites, and a lower gallic acid grafting rate is used to retain the carboxyl structure required for pectin to crosslink with calcium ions and to maintain its binding ability with the gallnut polyphenol interface layer.

[0077] In the preparation of tea tree oil-plant extract emulsion, tea tree oil, sophora flavescens extract, scutellaria baicalensis extract, tea saponin and soybean lecithin are added to an aqueous phase at pH 6.3 for emulsification.

[0078] In this embodiment, the solid mass ratio of Sophora flavescens extract, Scutellaria baicalensis extract and Galla chinensis polyphenol extract is 1:0.82:0.52.

[0079] In this embodiment, low-methoxylated citrus pectin with an esterification degree of 21.4% was used, and laccase-catalyzed grafting reaction was carried out according to a molar ratio of gallic acid to galacturonic acid units of 0.10:1. The grafting rate of gallic acid-grafted low-methoxylated citrus pectin was 11.6%.

[0080] In this embodiment, the dry basis addition of gallic acid-grafted low-methoxy citrus pectin is 0.40g, accounting for 4.0% of the tea tree oil mass.

[0081] In this embodiment, 0.40g of gallic acid-grafted low-methoxy citrus pectin was dissolved in 39.60g of deionized water to prepare a grafted pectin aqueous solution with a mass fraction of 1.0%, and then added to the emulsion forming a polyphenol interface layer according to the dropwise addition method in Example 1.

[0082] In this embodiment, the galacturonic acid content of the low-methoxy citrus pectin is calculated to be 80.0%, and combined with the degree of esterification of 21.4%, the molar amount of carboxyl groups that can participate in calcium crosslinking is 1.43 mmol.

[0083] Based on a calcium ion to carboxyl group molar ratio of 0.23:1, 3.3 mL of 0.1 mol / L calcium chloride solution was added dropwise to the system to induce cross-linking.

[0084] After calcium ion cross-linking, the resulting system forms a microcapsule-type plant extract compound tea tree oil bactericide encapsulated with tea tree oil, sophora flavescens extract and scutellaria baicalensis extract.

[0085] The bactericide obtained in this embodiment was tested and found to have an average particle size of 1.88 μm, a PDI of 0.268, and a tea tree oil encapsulation rate of 78.9%. After centrifugation at 3000 r / min for 30 min, a small amount of the bactericide floated to the surface without significant demulsification.

[0086] The bactericide obtained in this embodiment was placed in a pH 6.5 buffer solution for 24 hours, and the release of characteristic components of tea tree oil was measured to be 15.2%.

[0087] The bactericide obtained in this embodiment was placed in a pH 4.8 hydrochloric acid buffer and treated with pectin-degrading enzyme for 24 hours. The release of characteristic components of tea tree oil was measured to be 33.1%.

[0088] The bactericide obtained in this embodiment was placed in pH 4.8 oxalate buffer and treated with pectin-degrading enzyme for 8 h and 24 h, and the release of characteristic components of tea tree oil was measured to be 54.6% and 75.3%, respectively.

[0089] The bactericide obtained in this embodiment was placed in a pH 4.8 citrate buffer solution and treated with pectin-degrading enzyme for 24 hours. The release of characteristic components of tea tree oil was measured to be 69.4%.

[0090] High-performance liquid chromatography (HPLC) was used to detect oligogalacturonic acid fragments with a degree of polymerization of 6 to 12 in the release solution after co-treatment with oxalic acid and pectin degrading enzyme.

[0091] Using gray mold as the test object, and converting the fungicide obtained in this embodiment to the same effective addition amount according to the tea tree oil content, the inhibition rate of gray mold mycelial growth was measured to be 80.5%, and the inhibition rate of gray mold spore germination was 82.1%.

[0092] In a simulated experiment of gray mold disease on detached tomato leaves, the fungicide obtained in this embodiment was sprayed onto the leaf surface and then inoculated with a suspension of gray mold spores. The leaves were cultured at 25°C and relative humidity above 90% for 72 hours, and the diameter of the lesions was measured to be 8.7 mm.

[0093] The results of this embodiment show that, even when the amounts of plant extracts, polyphenol interface materials, gallic acid grafting rate, and calcium ion crosslinking ratio are all close to their lower limits, it is still possible to prepare a plant extract compound tea tree oil fungicide with microcapsule structure, low release under normal environment, release under the combined action of oxalic acid or citric acid and pectin degrading enzymes, and inhibition of gray mold.

[0094] Example 3 This embodiment illustrates that, even when the amount of grafted pectin added and the ratio of calcium ion crosslinking are close to the upper limit, it is still possible to prepare a plant extract compound tea tree oil bactericide with a calcium crosslinked pectin outer wall and responsive release ability.

[0095] Except for the parameter adjustments described below, all other extraction, concentration, drying, grafting, emulsification, interface layer formation, pectin deposition, calcium crosslinking, filtration, and detection methods in this embodiment are performed in accordance with Example 1.

[0096] In the preparation of plant extracts, Sophora flavescens extract was obtained by extraction with 70% ethanol aqueous solution, Scutellaria baicalensis extract was obtained by extraction with 70% ethanol aqueous solution, and Galla chinensis polyphenol extract was obtained by extraction with 50% ethanol aqueous solution.

[0097] Based on 10.0g of tea tree oil, 1.53g of Sophora flavescens extract, 2.17g of Scutellaria baicalensis extract and 0.92g of Galla chinensis polyphenol extract were weighed out, so that the mass ratio of tea tree oil to the total solids of Sophora flavescens extract and Scutellaria baicalensis extract was 10:3.7, and the amount of Galla chinensis polyphenol extract added was 9.2% of the mass of tea tree oil.

[0098] In this embodiment, the solid mass ratio of Sophora flavescens extract, Scutellaria baicalensis extract and Galla chinensis polyphenol extract is 1:1.42:0.6.

[0099] In this embodiment, the amount of gallnut polyphenol extract used is close to the upper limit of the scope defined by the present invention, which is used to verify that a stable interface layer can still be formed on the surface of the emulsion droplets even with a higher amount of polyphenol interface material.

[0100] In the preparation of gallic acid grafted low-methoxylated citrus pectin, low-methoxylated citrus pectin with an esterification degree of 34.1% was selected, and laccase-catalyzed grafting reaction was carried out according to a molar ratio of gallic acid to galacturonic acid units of 0.30:1.

[0101] The grafting rate of gallic acid-grafted low-methoxy citrus pectin was 23.4% as determined by the Folin-Ciocalteu method.

[0102] In this embodiment, a higher degree of esterification is used to reduce the density of calcium ion crosslinking, and a higher gallic acid grafting rate is used to enhance the binding ability between the grafted pectin and the gallnut polyphenol interface layer.

[0103] In the preparation of tea tree oil-plant extract emulsion, tea tree oil, sophora flavescens extract, scutellaria baicalensis extract, tea saponin and soybean lecithin are added to an aqueous phase at pH 6.7 for emulsification.

[0104] The mass ratio of tea saponin to soybean lecithin is 1:1.8.

[0105] In this embodiment, low-methoxylated citrus pectin with an esterification degree of 34.1% was used, and laccase-catalyzed grafting reaction was carried out according to a molar ratio of gallic acid to galacturonic acid units of 0.30:1. The grafting rate of gallic acid-grafted low-methoxylated citrus pectin was 23.4%.

[0106] In this embodiment, the dry basis amount of gallic acid-grafted low-methoxy citrus pectin is 1.20g, accounting for 12.0% of the tea tree oil mass.

[0107] In this embodiment, 1.20g of gallic acid-grafted low-methoxy citrus pectin was dissolved in 118.80g of deionized water to prepare a grafted pectin aqueous solution with a mass fraction of 1.0%, and then added to the emulsion forming a polyphenol interface layer according to the dropwise addition method in Example 1.

[0108] In this embodiment, the galacturonic acid content of the low-methoxy citrus pectin is calculated to be 80.0%, and combined with the degree of esterification of 34.1%, the molar amount of carboxyl groups that can participate in calcium crosslinking is 3.59 mmol.

[0109] Based on a calcium ion to carboxyl group molar ratio of 0.48:1, 17.2 mL of 0.1 mol / L calcium chloride solution was added dropwise to the system to induce cross-linking.

[0110] After calcium ion cross-linking, the resulting system forms a microcapsule-type plant extract compound tea tree oil bactericide encapsulated with tea tree oil, sophora flavescens extract and scutellaria baicalensis extract.

[0111] The bactericide obtained in this embodiment was tested and found to have an average particle size of 3.14 μm, a PDI of 0.243, a tea tree oil encapsulation rate of 84.6%, and no obvious stratification after centrifugation at 3000 r / min for 30 min.

[0112] The bactericide obtained in this embodiment was placed in a pH 6.5 buffer solution for 24 hours, and the release of characteristic components of tea tree oil was measured to be 8.6%.

[0113] The bactericide obtained in this embodiment was placed in a pH 4.8 hydrochloric acid buffer and treated with pectin-degrading enzyme for 24 hours. The release of characteristic components of tea tree oil was measured to be 24.2%.

[0114] The bactericide obtained in this embodiment was placed in pH 4.8 oxalate buffer and treated with pectin-degrading enzyme for 8 h and 24 h, and the release of characteristic components of tea tree oil was measured to be 48.9% and 70.1%, respectively.

[0115] The bactericide obtained in this embodiment was placed in a pH 4.8 citrate buffer solution and treated with pectin-degrading enzyme for 24 hours. The release of characteristic components of tea tree oil was measured to be 64.8%.

[0116] High-performance liquid chromatography (HPLC) was used to detect oligogalacturonic acid fragments with a degree of polymerization of 6 to 12 in the release solution after co-treatment with oxalic acid and pectin degrading enzyme.

[0117] Using gray mold as the test object, and converting the fungicide obtained in this embodiment to the same effective addition amount according to the tea tree oil content, the inhibition rate of gray mold mycelial growth was 78.8%, and the inhibition rate of gray mold spore germination was 80.4%.

[0118] In a simulated experiment of gray mold disease on detached tomato leaves, the fungicide obtained in this embodiment was sprayed onto the leaf surface and then inoculated with a suspension of gray mold spores. The leaves were cultured at 25°C and a relative humidity of over 90% for 72 hours, and the diameter of the lesions was measured to be 9.5 mm.

[0119] The results of this embodiment show that, even when the amounts of plant extracts, polyphenol interface materials, gallic acid grafting rate, and calcium ion crosslinking ratio are all close to their upper limits, it is still possible to prepare a plant extract compound tea tree oil fungicide with microcapsule structure, low release under normal environment, release under the combined action of oxalic acid or citric acid and pectin degrading enzymes, and inhibition of gray mold.

[0120] Comparative Example 1 This comparative example illustrates that when a gallic acid-grafted low-methoxy citrus pectin interface layer is not formed, the orderly deposition ability of the pectin on the outer side of the droplet and the integrity of the calcium-crosslinked pectin outer wall are affected.

[0121] This comparative example was prepared based on Example 1, except that the step of adding gallnut polyphenol extract was omitted and no polyphenol interface layer was formed.

[0122] In this comparative example, the amounts and emulsification conditions of Sophora flavescens extract, Scutellaria baicalensis extract, tea tree oil, tea saponin, and soybean lecithin were the same as in Example 1.

[0123] The gallic acid-grafted low-methoxy citrus pectin used in this comparative example is the same as that in Example 1. The determination and calculation methods for its degree of esterification, grafting rate, molar amount of galacturonic acid unit and molar amount of carboxyl group are all performed in accordance with Example 1.

[0124] In the comparative example, 60.0 g of a gallic acid-grafted low-methoxy citrus pectin aqueous solution (1.0% by mass) from Example 1 was directly added to the tea tree oil-plant extract emulsion, wherein the dry weight of the gallic acid-grafted low-methoxy citrus pectin was 0.60 g, and calcium ion crosslinking was performed according to 6.8 mL of 0.1 mol / L calcium chloride solution from Example 1.

[0125] The formulation obtained in this comparative example showed a small amount of flocculent aggregates during the addition of grafted pectin and calcium ion cross-linking, indicating that grafted pectin is difficult to deposit stably around the surface of the emulsion droplets when the gallnut polyphenol interface layer is lacking.

[0126] This comparative example was used to verify the effect of the gallnut polyphenol interface layer on the deposition of gallic acid-grafted low-methoxy citrus pectin, the formation of calcium-crosslinked pectin outer walls, and the stability of microcapsule structures.

[0127] Comparative Example 2 This comparative example illustrates the effect of low-methoxylated citrus pectin (not grafted with gallic acid) on the stability of the microcapsule outer wall and its responsive release capability.

[0128] This comparative example was prepared based on Example 1, except that ungrafted low-methoxy citrus pectin was used instead of gallic acid-grafted low-methoxy citrus pectin.

[0129] The degree of esterification of the low-methoxylated citrus pectin used in this comparative example was 28.4%, and the method for determining the degree of esterification was carried out in accordance with Example 1.

[0130] In this comparative example, the grafting rate of ungrafted low-methoxy citrus pectin was recorded as 0%.

[0131] In this comparative example, Sophora flavescens extract, Scutellaria baicalensis extract and Galla chinensis polyphenol extract were prepared according to the method of Example 1, and a tea tree oil-plant extract emulsion was formed according to the emulsification conditions of Example 1.

[0132] In this comparative example, gallnut polyphenol extract was added to form a polyphenol interface layer according to the method in Example 1. Then, 60.0 g of 1.0% (w / w) aqueous solution of ungrafted low-methoxy citrus pectin was added, wherein the dry weight of ungrafted low-methoxy citrus pectin was 0.60 g. Crosslinking was carried out by adding 0.1 mol / L calcium chloride solution at a molar ratio of calcium ions to carboxyl groups in ungrafted low-methoxy citrus pectin of 0.35:1.

[0133] In this comparative example, the molar ratio of calcium ions to carboxyl groups in low-methoxy citrus pectin was determined according to the method for calculating the molar amount of carboxyl groups in Example 1.

[0134] The formulation obtained in this comparative example can form a pectin-coated structure, but the binding stability between the outer wall of the pectin and the polyphenol interface layer is lower than that in Example 1.

[0135] This comparative example was used to verify the effects of gallic acid grafting structure on pectin deposition stability, calcium cross-linked pectin outer wall integrity, and oxalic acid-added pectin-degrading enzyme response release capacity.

[0136] Comparative Example 3 This comparative example illustrates the effect of pectin esterification degree exceeding the limits defined in this invention on the formation of the outer wall of calcium-crosslinked pectin and its responsive release capability.

[0137] This comparative example was prepared based on Example 1, except that gallic acid-grafted citrus pectin with an esterification degree of 44.6% was used to prepare the citrus pectin.

[0138] In this comparative example, the degree of esterification of citrus pectin was determined using the acid-base titration method of Example 1.

[0139] In this comparative example, the molar ratio of gallic acid to galacturonic acid units in citrus pectin was 0.20:1.

[0140] The grafting rate of gallic acid-grafted citrus pectin obtained in this comparative example was 18.1%, and the grafting rate was determined and calculated according to Example 1.

[0141] In this comparative example, a tea tree oil-plant extract emulsion and a gallnut polyphenol interface layer were prepared according to the method of Example 1, and gallic acid-grafted citrus pectin was deposited on the outside of the polyphenol interface layer.

[0142] In this comparative example, 0.60 g of gallic acid-grafted citrus pectin was prepared into 60.0 g of a 1.0% aqueous solution, and cross-linking was carried out by adding 0.1 mol / L calcium chloride solution according to the molar ratio of calcium ions to carboxyl groups in gallic acid-grafted citrus pectin of 0.35:1.

[0143] The formulation obtained in this comparative example showed increased particle size and decreased dispersibility after cross-linking, indicating that the unesterified carboxyl group segments that can participate in calcium ion cross-linking are insufficient in the highly esterified pectin.

[0144] This comparative example was used to verify the effect of the esterification degree range of low-methoxy citrus pectin on the formation of calcium-crosslinked pectin outer wall, general environmental stability, and pectin-degrading enzyme cleavage response.

[0145] Comparative Example 4 This comparative example is used to illustrate the structural and functional differences between ordinary tea tree oil pectin microcapsules and the formulation of the present invention.

[0146] This comparative example uses only tea tree oil, low-methoxylated citrus pectin, and calcium chloride to prepare tea tree oil microcapsules.

[0147] This comparative example does not include Sophora flavescens extract, Scutellaria baicalensis extract, or Galla chinensis polyphenol extract, does not prepare gallic acid-grafted low-methoxylated citrus pectin, and does not form a polyphenol interface layer.

[0148] The degree of esterification of the low-methoxylated citrus pectin used in this comparative example was 28.5%, and the method for determining the degree of esterification was carried out in accordance with Example 1.

[0149] In this comparative example, tea tree oil was emulsified and then 60.0 g of a 1.0% (w / w) aqueous solution of low-methoxylated citrus pectin was added, of which the dry weight of low-methoxylated citrus pectin was 0.60 g. 0.1 mol / L calcium chloride solution was added at a molar ratio of calcium ions to carboxyl groups in the low-methoxylated citrus pectin of 0.35:1 to crosslink and form a common pectin coating structure.

[0150] In this comparative example, the molar ratio of calcium ions to carboxyl groups in low-methoxy citrus pectin was 0.35:1, and the molar amount of carboxyl groups was calculated according to Example 1.

[0151] This comparative example was used to verify the effects of the absence of the compound system of Sophora flavescens extract and Scutellaria baicalensis extract, the gallnut polyphenol interface layer, and the gallic acid-grafted pectin outer wall on the stability of ordinary tea tree oil pectin microcapsules, the release of organic acid-pectin degrading enzymes under coexisting conditions, and the inhibitory effect on gray mold.

[0152] Comparative Example 5 This comparative example is used to illustrate the difference between the simple emulsified compound preparation of tea tree oil and plant extracts and the microcapsule preparation of this invention.

[0153] In this comparative example, tea tree oil, sophora flavescens extract, scutellaria baicalensis extract, gallnut polyphenol extract, tea saponin, and soybean lecithin were weighed according to the proportions specified in Example 1.

[0154] In this comparative example, the solid content of the Sophora flavescens extract, Scutellaria baicalensis extract, and Galla chinensis polyphenol extract was determined according to the solid content determination method described in Example 1.

[0155] In this comparative example, the above materials were added to 80.0g of an aqueous phase at pH 6.5 and emulsified under the high-speed shear conditions of Example 1.

[0156] This comparative example does not contain gallic acid-grafted low-methoxy citrus pectin, does not contain calcium chloride solution, and does not form a calcium cross-linked pectin outer wall.

[0157] The formulation obtained in this comparative example is a common emulsified compound system of tea tree oil, sophora flavescens extract, scutellaria baicalensis extract and gallnut polyphenol extract.

[0158] This comparative example is used to verify that simple emulsification and compounding of tea tree oil and plant extracts cannot replace the orderly construction of the polyphenol interface layer, the gallic acid-grafted low-methoxy citrus pectin deposition layer, and the calcium-crosslinked pectin outer wall.

[0159] Experimental Example 1 This experimental example is used to evaluate the microcapsule structure formation, tea tree oil encapsulation, and storage stability of the formulations obtained in Examples 1 to 3 and Comparative Examples 1 to 5.

[0160] The formulations obtained in Examples 1 to 3 and Comparative Examples 1 to 5 were diluted 100 times with deionized water and the average particle size and PDI were measured.

[0161] The preparations obtained in Examples 1 to 3 and Comparative Examples 1 to 5 were used to determine the total content and free content of tea tree oil by gas chromatography, with terpinen-4-ol in tea tree oil as the characteristic component, and the encapsulation rate of tea tree oil was calculated.

[0162] Take the formulations obtained from Examples 1 to 3 and Comparative Examples 1 to 5, centrifuge them at 3000 r / min for 30 min, and observe whether layering, flocculation or demulsification occurs.

[0163] The formulations obtained in Examples 1 to 3 and Comparative Examples 1 to 5 were heat-stored at 54°C for 14 days, and the changes in appearance stability were observed.

[0164] Each experiment was performed in parallel three times, and the average value of the results was taken.

[0165] Table 1: Results of Structural and Stability Testing for Examples and Comparative Examples In the table above, structural observation can be performed by using an optical microscope, laser confocal microscope, or transmission electron microscope to observe the continuity of the outer coating layer of the droplets, and by combining the particle size, PDI, and centrifugal stability to determine whether the polyphenol interface layer and the calcium cross-linked pectin outer wall have been formed.

[0166] Note: For Examples 1 to 3 and Comparative Examples 1 to 4, the values ​​in the table represent the encapsulation rate of tea tree oil.

[0167] Comparative Example 5 did not form capsule walls. The values ​​in the table represent the tea tree oil retention rate in the emulsion system after centrifugation.

[0168] As shown in Table 1, all three examples were able to form microcapsule structures with relatively concentrated particle size distribution, indicating that the present invention is feasible under preferred parameters, lower limit parameters, and higher limit parameters.

[0169] The average particle size of Example 1 was 2.36 μm, the PDI was 0.214, and the tea tree oil encapsulation rate was 86.7%, indicating that the microcapsules formed under the preferred conditions had relatively intact outer walls and good encapsulation effect.

[0170] Comparative Example 1 did not form a gallnut polyphenol interface layer, but its average particle size increased to 4.87 μm, PDI increased to 0.471, and tea tree oil encapsulation rate decreased to 62.5%, indicating that the gallnut polyphenol interface layer can guide the orderly deposition of gallic acid-grafted low-methoxy citrus pectin on the surface of the latex droplets.

[0171] Comparative Example 2 used ungrafted low-methoxyl citrus pectin, and its tea tree oil encapsulation rate was 70.8%, which was lower than 86.7% in Example 1, indicating that the gallic acid grafting structure can enhance the binding stability between the low-methoxyl citrus pectin and the polyphenol interface layer.

[0172] Comparative Example 3 used citrus pectin with an esterification degree of 44.6%, and its PDI increased to 0.506, while the encapsulation rate of tea tree oil decreased to 58.9%, indicating that when the pectin esterification degree is too high, there are insufficient carboxyl segments that can participate in calcium ion crosslinking.

[0173] Comparative Example 4 is a common tea tree oil pectin microcapsule, whose tea tree oil encapsulation rate and thermal storage stability are lower than those of Example 1, indicating that the common pectin coating structure cannot replace the polyphenol interface layer and grafted pectin outer wall in this invention.

[0174] Comparative Example 5 is a simple emulsified compound system with a PDI of 0.584 and a tea tree oil encapsulation rate of 39.6%, indicating that it is difficult to obtain a stable encapsulation structure without the formation of a calcium cross-linked pectin outer wall.

[0175] Table 1 shows that the stable microcapsule structure obtained in this invention is derived from the continuous construction of tea tree oil droplets, a polyphenol interface layer, a gallic acid-grafted low-methoxy citrus pectin deposition layer, and a calcium-crosslinked pectin outer wall, and is combined with Sophora flavescens extract and Scutellaria baicalensis extract to form a bactericidal active system.

[0176] Experiment Example 2 This experimental example is used to evaluate the release behavior of Examples 1 to 3 and Comparative Examples 1 to 5 under different simulated environments.

[0177] In this experiment, a pH 6.5 buffer solution was used as the medium to simulate a normal leaf surface environment.

[0178] In this experiment, a pH 6.5 buffer solution with pectin-degrading enzyme was used as a control medium for enzyme treatment alone.

[0179] In this experiment, a pH 4.8 hydrochloric acid buffer solution with pectin-degrading enzyme was used as a simulated medium for a common low-pH enzyme-added environment.

[0180] In this experimental example, pH 4.8 oxalate buffer was set as the treatment medium for calcium complex organic acids alone.

[0181] In this experiment, a pH 4.8 oxalate buffer solution with pectin-degrading enzyme was used as the medium for the co-action of oxalate and pectin-degrading enzyme.

[0182] In this experimental example, a pH 4.8 citrate buffer solution was set up as a separate treatment medium for another type of calcium complex organic acid.

[0183] In this experiment, a pH 4.8 citrate buffer solution with pectin-degrading enzyme was used as the medium for the co-action of citrate and pectin-degrading enzyme.

[0184] The pectin-degrading enzyme used was a mixed enzyme system of polygalacturonase and pectin lyase, with polygalacturonase at a concentration of 0.5 U / mL and pectin lyase at a concentration of 0.2 U / mL.

[0185] Each formulation was placed in a dialysis bag and then placed in the release medium described above. The mixture was then shaken at 25°C and 100 rpm to release the formulation.

[0186] The release solution was collected at 8h and 24h, and the release rate of tea tree oil was determined using terpinen-4-ol as the characteristic component.

[0187] The release solution after treatment with oxalic acid and pectin-degrading enzyme was subjected to ultrafiltration to remove macromolecular substances, and then high performance liquid chromatography was used to detect oligogalacturonic acid fragments with a degree of polymerization of 6 to 12.

[0188] Each experiment was performed in parallel three times, and the average value of the results was taken.

[0189] Table 2: Release results of the examples and comparative examples under different simulated environments As shown in Table 2, the release rate of pectin-degrading enzyme in Example 1 was 16.7% after 24 hours under pH 6.5 + pectin-degrading enzyme conditions, which was significantly lower than 81.5% under pH 4.8 oxalic acid + pectin-degrading enzyme conditions. This indicates that pectin-degrading enzyme alone is insufficient to fully release the calcium-crosslinked pectin outer wall.

[0190] In Example 1, the release rate after 24 hours under oxalic acid treatment alone at pH 4.8 was 26.1%, which was lower than the 81.5% under the condition of oxalic acid + pectin degrading enzyme at pH 4.8. This indicates that even after oxalic acid weakens the calcium cross-linking structure, pectin degrading enzyme is still needed to cleave the pectin main chain to achieve high release.

[0191] In Example 1, the release rate after 24 hours under citric acid treatment alone at pH 4.8 was 23.9%, which was lower than the 74.6% under the condition of citric acid + pectin degrading enzyme at pH 4.8. This indicates that citric acid can also act as a complex calcium-type organic acid to weaken the calcium cross-linking structure and promote release together with pectin degrading enzyme.

[0192] The above results indicate that the release process of the present invention includes two steps: the weakening of the calcium cross-linking structure by calcium complex organic acids and the cleavage of the pectin backbone by pectin-degrading enzymes.

[0193] As shown in Table 2, the release amount of Example 1 in pH 6.5 buffer solution was 11.8% in 24 hours, indicating that it has a low release level under normal conditions.

[0194] In Example 1, the 24-hour release of pectin-degrading enzyme under pH 4.8 hydrochloric acid and pectin-degrading enzyme conditions was 29.4%, while the 24-hour release of pectin-degrading enzyme under the same pH oxalic acid and pectin-degrading enzyme conditions was 81.5%, indicating that the weakening of the calcium cross-linking structure by oxalic acid can improve the cutting effect of pectin-degrading enzyme on the outer wall of pectin.

[0195] In Example 1, the release rate after 24 hours under the conditions of citric acid and pectin-degrading enzyme at pH 4.8 was 74.6%, indicating that citric acid, as an organic acid that can bind to calcium ions, can also weaken the calcium-crosslinked pectin outer wall and promote release.

[0196] In Example 1, the release rate after 24 hours in pH 4.8 oxalate buffer without the addition of pectin-degrading enzyme was 26.1%, indicating that the release was limited when only oxalate weakened the calcium cross-linking structure.

[0197] In Example 1, the release rate reached 63.7% after 8 hours under the conditions of oxalic acid and pectin-degrading enzyme at pH 4.8, indicating that there is a continuous synergistic effect between oxalic acid weakening the calcium cross-linking structure and pectin-degrading enzyme cleaving the pectin backbone.

[0198] The 24-hour release of Comparative Example 1 in pH 6.5 buffer was 22.7%, which was higher than the 11.8% of Example 1, indicating that the lack of the gallnut polyphenol interface layer reduces the stability of the outer wall in normal environment and leads to an increase in premature release.

[0199] Comparative Example 2 showed a release rate of 61.8% after 24 hours under oxalic acid and pectin-degrading enzyme conditions at pH 4.8, which was lower than the 81.5% of Example 1. This indicates that ungrafted pectin cannot fully form a calcium-crosslinked pectin outer wall that is both stable and responsive.

[0200] Comparative Example 3 showed a release rate of 43.7% after 24 hours under oxalic acid and pectin-degrading enzyme conditions at pH 4.8, indicating that both the calcium cross-linking structure and the enzymatic response structure were affected when the pectin esterification was too high.

[0201] The small difference in release amount in Comparative Example 5 across different release media indicates that the simple emulsified compound system lacks the release difference between low release in normal environments and high release under conditions of coexistence of organic acids and pectin-degrading enzymes.

[0202] In Examples 1 to 3, DP6-12 oligogalacturonic acid fragments were detected in the release solution after treatment with oxalic acid and pectin-degrading enzyme, indicating that the pectin backbone in the outer wall of calcium-crosslinked pectin was cleaved by enzymes.

[0203] Table 2 shows that the release mechanism of the present invention includes the weakening of the calcium cross-linking structure by calcium complex organic acids, followed by the cleavage of the pectin backbone by pectin-degrading enzymes and the promotion of the release of active ingredients.

[0204] Experimental Example 3 This experiment was used to evaluate the inhibitory effects of Examples 1 to 3 and Comparative Examples 1 to 5 on Botrytis cinerea.

[0205] This experiment used Botrytis cinerea, a gray mold, as the test subject. The gray mold was obtained by activating a laboratory-preserved strain and cultured on PDA medium at 25°C for 3 days before use.

[0206] Each formulation was adjusted to the same effective addition amount of 500 mg / L based on the tea tree oil content.

[0207] The mycelial growth inhibition experiment was conducted using the mycelial growth rate method. A 5 mm diameter Botrytis cinerea fungal cake was inoculated into the center of a PDA plate containing the corresponding preparation, and the colony diameter was measured after incubation at 25°C for 72 h.

[0208] The mycelial growth inhibition rate is calculated using the following formula: Mycelial growth inhibition rate = [Diameter of blank control colonies - Diameter of treatment group colonies] / Diameter of blank control colonies × 100%.

[0209] In the spore germination inhibition experiment, the gray mold spore suspension was adjusted to 1.0 × 10⁻⁶. 5 The spores were mixed at a concentration of 1 spore per mL with an equal volume of each formulation and incubated at 25°C for 8 hours. The spore germination was then counted under a microscope.

[0210] The spore germination inhibition rate was calculated using the following formula: Spore germination inhibition rate = [Spore germination rate of blank control group - spore germination rate of treatment group] / spore germination rate of blank control group × 100%.

[0211] Each treatment group was set up with 3 parallel replicates, and the results are expressed as mean ± standard deviation.

[0212] In the experiment on detached tomato leaves, functional tomato leaves with uniform growth and no mechanical damage were selected, with 10 leaves set in each group.

[0213] Each formulation was sprayed on both sides of the leaves after being converted to a tea tree oil content of 500 mg / L, with a spraying amount of 1.0 mL per leaf.

[0214] After the leaf surface has air-dried naturally, inoculate the center of the leaf with 10 μL of a 1.0 × 10⁻⁶ solution. 5 A suspension of gray mold spores per mL.

[0215] After inoculation, the leaves were cultured at 25℃ and relative humidity above 90% for 72 hours, and the diameter of the lesions was measured using the cross-cross method.

[0216] In the oxalic acid and pectin-degrading enzyme pretreatment experiment, each preparation was first placed in a pH 4.8 oxalic acid buffer and pectin-degrading enzyme system for 8 hours, and then used in the mycelial growth inhibition experiment.

[0217] In the plant defense auxiliary verification experiment, after each preparation was sprayed on the surface of tomato leaves, samples were taken to measure PAL activity and POD activity, and the enzyme activity of the blank treatment group was taken as 1.00 times.

[0218] Each experiment was performed in parallel three times, and the average value of the results was taken.

[0219] Table 3: Inhibitory Effects of Examples and Comparative Examples on Botrytis cinerea As shown in Table 3, Example 1 showed an 86.2% inhibition rate on mycelial growth of gray mold and an 88.7% inhibition rate on spore germination. The diameter of the lesion on the detached tomato leaf was 6.9 mm.

[0220] Under the pretreatment conditions of oxalic acid and pectin-degrading enzyme, the mycelial growth inhibition rate of Example 1 was 93.5%, which was higher than 86.2% under the untreated conditions. Combined with the release experiment results, it is shown that the coexistence of organic acid and pectin-degrading enzyme can promote the release of active ingredients in the preparation and is beneficial to improve the in vitro antibacterial performance.

[0221] Examples 2 and 3 represent the lower and higher limits of parameters, respectively, with mycelial growth inhibition rates of 80.5% and 78.8%, respectively, demonstrating that the present invention can achieve effective bactericidal effects within the scope of the claims.

[0222] The mycelial growth inhibition rate of Comparative Example 1 was 61.7%, which was lower than that of Example 1 (86.2%), indicating that the lack of a gallnut polyphenol interface layer would affect the stability of the microcapsules and the control of the release of the active ingredients.

[0223] The mycelial growth inhibition rate of Comparative Example 2 was 67.3%, which was lower than that of Example 1 (86.2%), indicating that ungrafted low-methoxy citrus pectin is difficult to replace gallic acid-grafted low-methoxy citrus pectin.

[0224] The mycelial growth inhibition rate of Comparative Example 3 was 54.8%, and the diameter of the lesion on the detached tomato leaf was 18.2 mm, indicating that excessive pectin esterification reduces the quality of calcium cross-linked outer wall formation and affects the actual control efficacy.

[0225] The mycelial growth inhibition rate of Comparative Example 4 was 59.5%, which was lower than 86.2% in Example 1, indicating that ordinary tea tree oil microcapsules cannot achieve the comprehensive effect of the tea tree oil and plant extract compound system and the response outer wall of this invention.

[0226] The mycelial growth inhibition rate of Comparative Example 5 was 68.1%, and the diameter of the lesion on the detached tomato leaf was 12.7 mm. This indicates that although the simple emulsified compound has a certain antibacterial effect, it still lacks the stable release and leaf protection effect brought by the microcapsule structure of this invention.

[0227] The inhibition rates of Comparative Examples 1 to 4 after pretreatment with oxalic acid and pectin-degrading enzyme were all lower than those of Example 1, indicating that when there is a lack of polyphenol interface layer, gallic acid grafting structure or pectin with a suitable degree of esterification, the formulation is difficult to form a stable and responsive calcium-crosslinked pectin outer wall.

[0228] Although Comparative Example 5 contains tea tree oil and plant extracts, it lacks the calcium-crosslinked pectin outer wall, and its lesion diameter is still larger than that of Example 1, indicating that simple emulsification and compounding cannot replace the microcapsule structure of the present invention.

[0229] Table 3 shows that the present invention stabilizes the active ingredients through the microcapsule structure and enhances the release of active ingredients under simulated conditions of oxalic acid and pectin-degrading enzymes, thereby improving the inhibitory effect on gray mold and the protective effect on tomato leaves.

[0230] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a plant extract compounded with tea tree oil bactericide, characterized in that, include: S1. Prepare extracts of Sophora flavescens, Scutellaria baicalensis, and Galla chinensis polyphenols, and determine the dosage of the three extracts according to a solid mass ratio of 1:(0.8-1.5):(0.3-0.8). S2, using low-methoxylated citrus pectin and gallic acid as raw materials, gallic acid-grafted low-methoxylated citrus pectin is prepared, wherein the degree of esterification of the low-methoxylated citrus pectin is 20%–35%, and the grafting rate of the gallic acid-grafted low-methoxylated citrus pectin is 10%–25%; S3, tea tree oil, sophora flavescens extract, scutellaria baicalensis extract, tea saponin and soybean lecithin were added to an aqueous phase with a pH of 6.2 to 6.8 and emulsified to obtain a tea tree oil-plant extract emulsion; S4, add gallnut polyphenol extract to tea tree oil-plant extract emulsion, so that gallnut polyphenols are bound to the surface of emulsion droplets to form a polyphenol interface layer; S5, add gallic acid-grafted low-methoxy citrus pectin to the emulsion that forms a polyphenol interface layer, so that the gallic acid-grafted low-methoxy citrus pectin is deposited on the outside of the polyphenol interface layer. S6, add calcium chloride solution to crosslink calcium ions with carboxyl groups in gallic acid-grafted low-methoxy citrus pectin, forming a calcium crosslinked pectin outer wall on the outside of the droplets, to obtain a microcapsule-type plant extract compound tea tree oil bactericide. The microcapsule-type plant extract compound tea tree oil bactericide includes tea tree oil-containing emulsion droplets, a polyphenol interface layer, and a calcium cross-linked pectin outer wall, and contains Sophora flavescens extract and Scutellaria baicalensis extract. The polyphenol interface layer is bonded to the surface of the tea tree oil-containing emulsion droplets, and the calcium cross-linked pectin outer wall is located outside the polyphenol interface layer. The outer wall of the calcium-crosslinked pectin includes a crosslinked structure formed by grafting gallic acid onto carboxyl groups and calcium ions in low-methoxy citrus pectin. The crosslinked structure can be weakened in the presence of oxalic acid or citric acid, and the pectin backbone in the outer wall of the calcium-crosslinked pectin can be cleaved in the presence of pectin-degrading enzymes.

2. The method for preparing a plant extract compound tea tree oil bactericide as described in claim 1, characterized in that, In S1, the Sophora flavescens extract is obtained by extraction with 60%–70% ethanol aqueous solution, the Scutellaria baicalensis extract is obtained by extraction with 50%–70% ethanol aqueous solution, and the Galla chinensis polyphenol extract is obtained by extraction with 30%–50% ethanol aqueous solution.

3. The method for preparing a plant extract compound tea tree oil bactericide as described in claim 1, characterized in that, In S1, the solid mass ratio of the Sophora flavescens extract, Scutellaria baicalensis extract and Galla chinensis polyphenol extract is 1:(1-1.2):(0.4-0.6).

4. The method for preparing a plant extract compound tea tree oil bactericide as described in claim 1, characterized in that, In S2, the molar ratio of gallic acid to galacturonic acid units in the low-methoxy citrus pectin is (0.10-0.30):

1.

5. The method for preparing a plant extract compound tea tree oil bactericide as described in claim 1, characterized in that, In S2, the gallic acid-grafted low-methoxy citrus pectin is prepared by a laccase-catalyzed grafting reaction.

6. The method for preparing a plant extract compound tea tree oil bactericide as described in claim 1, characterized in that, In S3, the mass ratio of tea saponin to soybean lecithin is 1:(0.5-2).

7. The method for preparing a plant extract compound tea tree oil bactericide as described in claim 1, characterized in that, In S3, the mass ratio of the tea tree oil to the total solids of the sophora flavescens extract and scutellaria baicalensis extract is 10:(1-4).

8. The method for preparing a plant extract compound tea tree oil bactericide as described in claim 1, characterized in that, In S4, the amount of gallnut polyphenol extract added is 3% to 10% of the mass of tea tree oil.

9. The method for preparing a plant extract compound tea tree oil bactericide as described in claim 1, characterized in that, In S5, the dry basis addition amount of the gallic acid-grafted low-methoxy citrus pectin is 4% to 12% of the mass of tea tree oil.

10. The method for preparing a plant extract compound tea tree oil bactericide as described in claim 1, characterized in that, In S6, the molar ratio of calcium ions to carboxyl groups in the gallic acid-grafted low-methoxy citrus pectin is (0.20–0.50):1.

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