Antibacterial agent of guijiu mango peel extract and preparation method thereof

Abstract

This invention relates to an antibacterial agent derived from the peel of *Guiqi* mango and its preparation method, belonging to the field of natural product nanoparticle technology. The method aims to solve the technical problem of how to simultaneously achieve efficient co-extraction of water-soluble and lipid-soluble antibacterial components in a continuous process, and to immediately stabilize easily oxidized components, as well as to construct a high-encapsulation-rate nanoparticle antibacterial agent. The key technical points are: after mixing dried mango peel powder, β-cyclodextrin, and deionized water, a high-voltage pulsed electric field is used to simultaneously achieve cell wall disruption extraction and in-situ molecular inclusion of active ingredients; subsequently, the obtained inclusion extract is mixed with chitosan hydrochloride solution, and then ion cross-linked by adding sodium tripolyphosphate aqueous solution to construct composite nanoparticles; finally, after ethanol aging, centrifugation purification, and redispersing in buffer solution, the final product in nanoparticle form is obtained. The antibacterial agent prepared by this method can be used in the fields of preservation and antibacterial activity.

Description

Technical Field

[0001] This invention belongs to the field of natural product nano-preparation technology, specifically relating to an antibacterial agent of Guiqi mango peel extract and its preparation method. Background Technology

[0002] Currently, obtaining antibacterial active substances from plant materials typically relies on a stepwise extraction and subsequent encapsulation process. For plant materials containing both water-soluble and fat-soluble antibacterial components, such methods require extraction using different solvent systems, or the addition of stabilization measures for fat-soluble and easily oxidized components after extraction. This stepwise operation not only makes the process cumbersome and time-consuming, but also makes it easy for some heat-sensitive or chemically unstable active ingredients to be lost or degraded during intermediate processing and transfer. Furthermore, treating extraction and encapsulation or nano-formulation as independent unit operations increases process complexity and control difficulty, making it difficult to achieve efficient, continuous, and immediate stability of active ingredients from release to encapsulation, potentially affecting the overall antibacterial efficacy and shelf-life stability of the final product. It is worth noting that the peel of the Guiqi mango, as a large byproduct generated during the processing of specialty agricultural products, is widely available, inexpensive, and rich in various active ingredients with antibacterial potential, such as polyphenols and flavonoids. Its high-value resource utilization has significant economic and social benefits. However, its complex composition, the presence of easily oxidized substances, and the inefficiencies of traditional step-by-step processing technology in terms of efficiency and component protection have hindered the development of its antibacterial products with high efficiency and high quality.

[0003] Based on this, the present invention provides a method for preparing an antibacterial agent from Guiqi mango peel extract, which aims to achieve efficient co-extraction, in-situ stabilization and nano-formulation of active ingredients simultaneously through an integrated continuous process, so as to overcome the above-mentioned technical defects. Summary of the Invention

[0004] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0005] Another objective of this invention is to provide a method for preparing an antibacterial agent from Guiqi mango peel extract. This method enables the efficient co-extraction of water-soluble and fat-soluble antibacterial active ingredients from Guiqi mango peel in a continuous process. It also utilizes β-cyclodextrin to achieve in-situ molecular inclusion of easily oxidized and degradable components to achieve immediate stability. Furthermore, it constructs composite nanoparticles through ion crosslinking technology, ultimately obtaining an antibacterial agent product with high encapsulation efficiency, good stability, and long-lasting sustained-release potential. This method effectively overcomes the problems of low efficiency, significant component loss, and complex operation associated with traditional stepwise processes.

[0006] To achieve these objectives and other advantages of the present invention, a method for preparing an antibacterial agent from Guiqi mango peel extract is provided, comprising the following steps: S1. Mix dried mango peel powder, β-cyclodextrin, and deionized water to obtain a solid-liquid mixture system; wherein the mass-to-volume ratio of mango peel powder to deionized water is 1g:15-20mL, and the mass concentration of β-cyclodextrin relative to deionized water is 3%-6%; place the solid-liquid mixture system under an electric field strength of 20-30kV / cm, a pulse width of 20-40μs, and a pulse frequency of 100-200Hz for 10-20min, and maintain the system temperature at 25-35℃ during the treatment; after the treatment, immediately transfer the mixture system to a stirred reactor and stir continuously at 35-45℃ and a rotation speed of 300-500r / min for 3-5h to obtain an extraction inclusion mixture; S2. Filter the extract-inclusion mixture obtained in step S1, collect the filtrate, and obtain a clear β-cyclodextrin inclusion extract; mix the β-cyclodextrin inclusion extract with chitosan hydrochloride solution at a volume ratio of 4-6:1 to obtain a second mixture; disperse the second mixture at 25-30℃ and a high-speed shearing speed of 10000-15000 r / min for 2-4 min. S3. Under continuous stirring at 800-1500 r / min, sodium tripolyphosphate aqueous solution is added dropwise to the second mixture after high-speed shear dispersion. The mass concentration of the sodium tripolyphosphate aqueous solution is 0.08-0.12%, and the volume of the added solution is 4-6% of the volume of the second mixture. After the addition is complete, stirring is continued at 25-30℃ for 40-80 min to obtain a nanoparticle suspension. S4. The nanoparticle suspension obtained in step S3 is mixed with a 50-70% (v / v) food-grade ethanol aqueous solution at a volume ratio of 10-15:1 and aged at 20-25℃ for 2-4 hours. After aging, the mixture is centrifuged at 8000-12000 r / min for 15-25 min, and the precipitate is collected. The precipitate is redispersed in a 0.01 mol / L citrate-sodium citrate buffer solution with a pH of 5.5-6.5 at a ratio of 1 g of precipitate to 20-30 mL of buffer solution. The solution is then filtered through a 0.22 μm filter membrane to remove bacteria, thus obtaining the antibacterial agent of Guiqi mango peel extract.

[0007] Dried mango peel powder was mixed with an aqueous solution of β-cyclodextrin and then treated with a high-voltage pulsed electric field. Under the influence of the electric field, the plant cell walls were rapidly disrupted, causing the simultaneous release of various water-soluble and lipid-soluble antibacterial active ingredients into the aqueous phase. During this process, the β-cyclodextrin molecules dissolved in the medium, with their hydrophobic cavities, could instantly encapsulate the released lipid-soluble and easily oxidized components, thus stabilizing unstable components during extraction. Subsequently, a clear inclusion extract was obtained by filtration and mixed with a chitosan hydrochloride solution, followed by high-speed shearing to form a homogeneous system. Under continuous stirring, an aqueous solution of sodium tripolyphosphate was added dropwise to this system. Ionic cross-linking occurred through the electrostatic interaction between chitosan and tripolyphosphate ions, gradually constructing composite nanoparticles that could simultaneously physically encapsulate water-soluble components and contain β-cyclodextrin inclusion complexes. Finally, aging was carried out using an aqueous ethanol solution to further compact the nanoparticle structure and remove unencapsulated free components. After centrifugation, the nanoparticles were collected and redispersed in a buffer solution to obtain a stable nanoparticle-based antibacterial agent.

[0008] Preferably, the chitosan hydrochloride solution is prepared by dissolving chitosan hydrochloride with a degree of deacetylation greater than 90% in deionized water, resulting in a chitosan hydrochloride solution with a mass concentration of 0.8-1.2%.

[0009] Preferably, the dried mango peel powder is obtained by pulverizing dried Guiqi mango peel and passing it through a sieve with a mesh size of 180-250μm.

[0010] Preferably, the specific process of mixing dried mango peel powder, β-cyclodextrin, and deionized water in step S1 is as follows: First, the β-cyclodextrin of the formula amount is mixed with deionized water accounting for 60-70% of the total water required by the formula at 25-35°C, and then sheared and dispersed at a high speed of 2000-3000 r / min for 3-5 minutes to form a uniform pre-dispersed slurry; then, the dried mango peel powder of the formula amount is added to the pre-dispersed slurry, and the remaining deionized water is added to the total water required by the mass-volume ratio, and then stirred and mixed at a speed of 400-600 r / min for 10-15 minutes to obtain the solid-liquid mixture system.

[0011] First, a portion of deionized water and β-cyclodextrin are subjected to high-speed shear mixing at a suitable temperature. This high shear force effectively breaks up the agglomeration of β-cyclodextrin powder, promoting thorough wetting and dispersion to form a uniform slurry with fully exposed active sites. Subsequently, dried mango peel powder is added to this pre-dispersed slurry, and gentle mixing is achieved by adding the remaining water and stirring at a moderate speed. This sequence of operations aims to ensure that the β-cyclodextrin medium is highly homogenized before contacting the plant material, thereby creating an optimal liquid-phase environment for the release and capture of active ingredients in subsequent steps. At the same time, the gentle mixing method avoids unnecessary premature damage to the mango peel cell structure due to excessive mechanical force, ensuring that subsequent electric field treatment can be carried out efficiently in a stable system with uniform material distribution and fully activated inclusion media.

[0012] Preferably, the specific process of filtration of the extract-inclusion mixture obtained in step S1 in step S2 is as follows: constant pressure filtration is performed using a double-layer filter material, wherein the double-layer filter material consists of an upper layer of qualitative filter paper and a lower layer of nylon microporous filter membrane, wherein the pore size of the qualitative filter paper is 10-15 μm and the pore size of the nylon microporous filter membrane is 1.0-1.5 μm; during the filtration process, the vacuum pressure is controlled within the range of -0.04 MPa to -0.06 MPa and kept constant.

[0013] This step employs a double-layer filter media with decreasing pore size from top to bottom for vacuum filtration. The upper layer, a qualitative filter paper with larger pores, first traps most of the solid residue and disperses fluid pressure, effectively preventing rapid clogging of the lower precision filter membrane. The lower layer, a nylon microporous filter membrane with smaller pores, further ensures that the filtrate achieves the required clarity. Simultaneously, by precisely controlling and maintaining the vacuum pressure within a relatively mild and constant range throughout the filtration process, an appropriate filtration rate is ensured while minimizing the severe fluid shear forces caused by excessive pressure or fluctuations. This reduces potential disturbance to the β-cyclodextrin inclusion complex structure already formed in the filtrate and minimizes non-specific adsorption loss of active ingredients on the filter media surface, ultimately achieving efficient, mild, and high-yield solid-liquid separation.

[0014] Preferably, the aging process at 20-25°C for 2-4 hours described in step S4 is carried out in a sealed container, which rotates horizontally around its central axis at a speed of 0.5-2 rpm continuously and at a constant speed.

[0015] During aging in a sealed container, the container is continuously rotated horizontally around its central axis at an extremely low speed. This slow rotation generates very gentle laminar flow disturbances within the system, sufficient to counteract the settling tendency of nanoparticles due to gravity, allowing all particles to maintain a uniform suspension state for an extended period. In this quasi-static mixing environment, ethanol diffuses smoothly and uniformly throughout the system, promoting synchronous and uniform dehydration, shrinkage, and densification of the nanoparticles. This process avoids aging differences caused by localized concentration inconsistencies or particle settling, and also mitigates the potential damage to the fragile nanostructures caused by strong mechanical shearing, thus contributing to obtaining a final product with a more uniform particle size distribution and superior dispersion stability.

[0016] Preferably, in step S3, when adding sodium tripolyphosphate aqueous solution dropwise to the second mixture under continuous stirring, a staged variable-speed dropping method is adopted, which is coordinated with the corresponding stirring speed: in the initial stage of dropping, the dropping speed is controlled at 0.5-0.8% of the volume of the second mixture per minute, while the stirring speed is maintained at 800-1000 r / min; when the dropping volume reaches 40%-50% of the total amount of sodium tripolyphosphate aqueous solution to be added, the dropping is entered into the middle stage, and the dropping speed is adjusted to 0.2%-0.4% of the volume of the second mixture per minute, while the stirring speed is increased to 1200-1500 r / min and maintained until the dropping is completed.

[0017] When adding sodium tripolyphosphate aqueous solution dropwise to the second mixture for ionic crosslinking, a staged variable-speed dropwise addition strategy was adopted, coordinated with the stirring speed. In the initial stage of addition, the crosslinking agent was added at a relatively fast rate while maintaining moderate stirring intensity, aiming to quickly form sufficient crosslinking points and initially construct the nanoparticle morphology. When about half of the required total amount had been added, the process transitioned to the middle stage, at which point the dropwise addition rate was significantly reduced while the stirring speed was simultaneously increased. Reducing the dropwise addition rate helps avoid excessive crosslinking and aggregation caused by excessively high local concentrations of the crosslinking agent; while increased stirring enhances the shear dispersion effect of the system, promoting a more rapid and uniform distribution of the newly added crosslinking agent, ensuring sufficient contact with the already formed particle surfaces, thereby guiding a more uniform and dense crosslinking network growth, ultimately obtaining a nanoparticle suspension with a more stable structure and a more concentrated particle size distribution.

[0018] Preferably, during the continuous stirring process described in step S1 for 3-5 hours, D-mannitol is added to the stirred reactor 1.5-2.5 hours after the start of stirring. The amount added is 15%-25% of the mass of β-cyclodextrin in the solid-liquid mixture, and the remaining stirring time is continued.

[0019] During the continuous stirring process in step S1, food-grade D-mannitol is added to the system after a certain period of stirring. This small molecule can reversibly bind to the cavities of β-cyclodextrin. Through this dynamic competition and replacement, it helps to displace pre-encapsulated, weakly bound water molecules or impurity molecules in the β-cyclodextrin cavities, thereby providing more usable cavity space and better binding sites for the active ingredient. This allows the hydrophobic antibacterial component released from mango peel to more fully embed into the cyclodextrin cavities and adjust to a more thermodynamically stable inclusion posture, thereby enhancing the stability and inclusion efficiency of the inclusion complex.

[0020] Preferably, after obtaining the extraction-inclusion mixture in step S1 and before filtration in step S2, an enzymatic hydrolysis and viscosity reduction step is added: a compound enzyme preparation is added to the extraction-inclusion mixture for enzymatic hydrolysis; the compound enzyme preparation is composed of pectinase and cellulase in a mass ratio of 2-3:1, and its addition amount is 0.02-0.05% of the total mass of the extraction-inclusion mixture; the compound enzyme preparation contains pectinase activity ≥50000U / g and cellulase activity ≥20000U / g; the process conditions for enzymatic hydrolysis are: under continuous stirring, the system temperature is controlled at 38-42℃, the pH value is 4.0-5.0, and the hydrolysis time is 20-40 minutes; after the enzymatic hydrolysis is completed, the mixture is rapidly heated to 85-90℃ and maintained at this temperature for 5-10 minutes to completely inactivate the enzyme, and then naturally cooled to 25-30℃ before proceeding to the filtration operation in step S2.

[0021] The mixture obtained after extraction and inclusion has a high viscosity due to the presence of dissolved plant cell wall polysaccharides. To reduce viscosity and ensure the efficiency and uniformity of subsequent operations, a complex enzyme preparation composed of pectinase and cellulase is added, and the mixture is continuously stirred under mild acidic conditions. This complex enzyme specifically catalyzes the hydrolysis of high molecular weight pectin and some fibrous impurities that cause increased viscosity, converting them into low molecular weight fragments, thereby significantly reducing the system viscosity. Due to the high selectivity of enzymatic hydrolysis, this process does not affect the structure of the already formed β-cyclodextrin inclusion complex or the hydrophobic active ingredients encapsulated therein. After enzymatic hydrolysis, the enzyme protein is completely and rapidly inactivated by rapid heating and short-term holding to avoid residual enzyme activity interfering with subsequent ionic cross-linking processes. The inactivated enzyme protein and other soluble degradation products can be effectively removed in subsequent filtration and ethanol aging purification steps.

[0022] The present invention also provides a Guiqi mango peel extract antibacterial agent, which is prepared by the above-mentioned preparation method of Guiqi mango peel extract antibacterial agent.

[0023] The present invention has at least the following beneficial effects: The preparation method provided by this invention achieves integrated continuous operation of extraction, stabilization, and nano-formulation, simplifying the process flow, improving efficiency, and reducing the loss of active ingredients. By leveraging a high-voltage pulsed electric field in synergistically with the in-situ inclusion of β-cyclodextrin, both water-soluble and lipid-soluble antibacterial components can be efficiently co-extracted, and easily oxidized and degradable components can be stabilized immediately, thereby improving the overall retention rate and stability of active substances in the final product. Furthermore, the composite nanoparticle structure constructed through ion crosslinking and subsequent optimization steps such as ethanol aging collectively endow the final antibacterial agent with excellent encapsulation efficiency, redispersibility, and potential sustained-release properties, providing convenience for practical applications.

[0024] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Detailed Implementation

[0025] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0026] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0027] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0028] Example 1 A method for preparing an antibacterial agent from mango peel extract (such as *Gynostemma pentaphyllum*) includes the following steps: S1. Dried mango peel powder, β-cyclodextrin, and deionized water were mixed to obtain a solid-liquid mixture system. The mass-to-volume ratio of mango peel powder to deionized water was 1 g:17.5 mL, and the mass concentration of β-cyclodextrin relative to deionized water was 4.5% (i.e., 4.5 g of β-cyclodextrin was added per 100 mL or 100 g of deionized water). The solid-liquid mixture system was placed in a high-voltage pulsed electric field treatment chamber and treated for 15 min under the conditions of an electric field strength of 25 kV / cm, a pulse width of 30 μs, and a pulse frequency of 150 Hz. During the treatment, the system temperature was maintained at 30 °C. After the treatment, the mixture system was immediately transferred to a stirred reactor and stirred continuously for 4 h at 40 °C and a rotation speed of 400 r / min to obtain the extraction inclusion mixture. S2. Filter the extract-inclusion mixture obtained in step S1, collect the filtrate, and obtain a clear β-cyclodextrin inclusion extract; mix the β-cyclodextrin inclusion extract with chitosan hydrochloride solution at a volume ratio of 5:1 to obtain a second mixture; and shear the second mixture at 28°C and a speed of 12500 r / min for 3 min. S3. Under continuous stirring, sodium tripolyphosphate aqueous solution is added dropwise to the second mixture after high-speed shear dispersion. The mass concentration of the sodium tripolyphosphate aqueous solution is 0.10%, and the volume of the added solution is 5% of the volume of the second mixture. After the addition is completed, stirring is continued at 28°C for 60 min to obtain a nanoparticle suspension. S4. The nanoparticle suspension obtained in step S3 is mixed with a 60% (v / v) food-grade ethanol aqueous solution at a volume ratio of 12.5:1 and aged at 22℃ for 3 hours. After aging, the mixture is centrifuged at 10000 r / min for 20 minutes and the precipitate is collected. The wet precipitate collected by centrifugation is redispersed in a 0.01 mol / L citrate-sodium citrate buffer solution with a pH of 6.0 and a molar ratio of citric acid to sodium citrate of 1:2 at a ratio of 1 g to 25 mL. Then, the solution is filtered and sterilized using a 0.22 μm pore size filter membrane under positive pressure to obtain the antibacterial agent of Guiqi mango peel extract.

[0029] The chitosan hydrochloride solution was prepared by dissolving chitosan hydrochloride with a molecular weight range of 10-50 kDa and a degree of deacetylation greater than 90% in deionized water. The dissolution conditions were 25±5℃ and stirring at 500-800 r / min until completely dissolved, resulting in a final solution mass concentration of 1.0%. The pH value of the 1.0% chitosan hydrochloride solution was determined to be 5.2±0.3.

[0030] The dried mango peel powder is obtained by drying Guiqi mango peel with hot air (60~70℃) until the moisture content is ≤5.0%, and then pulverizing it to pass through a sieve with a pore size of 215μm.

[0031] The specific process of mixing dried mango peel powder, β-cyclodextrin, and deionized water in step S1 is as follows: First, the β-cyclodextrin of the formula amount is mixed with deionized water accounting for 65% of the total water volume required by the formula at 30°C, and then sheared and dispersed at a high speed of 2500 r / min for 4 minutes to form a uniform pre-dispersed slurry; then, the dried mango peel powder of the formula amount is added to the pre-dispersed slurry, and the remaining deionized water is added to the total water volume required by the mass-volume ratio, and then stirred and mixed at a speed of 500 r / min for 12.5 minutes to obtain the solid-liquid mixture system.

[0032] The specific process of filtration of the extract and inclusion mixture obtained in step S1 in step S2 is as follows: constant pressure filtration is performed using a double-layer filter material, which consists of an upper layer of qualitative filter paper and a lower layer of nylon microporous filter membrane. The pore size of the qualitative filter paper is 12.5 μm, and the pore size of the nylon microporous filter membrane is 1.25 μm. During the filtration process, the vacuum pressure is controlled at -0.05 MPa and kept constant.

[0033] In step S3, when adding sodium tripolyphosphate aqueous solution dropwise to the second mixture under continuous stirring, a staged variable-speed dropping method is adopted, which is coordinated with the corresponding stirring speed: in the initial stage of dropping, the dropping speed is controlled at 0.65% of the volume of the second mixture per minute, while the stirring speed is maintained at 900 r / min; when the dropping volume reaches 45% of the total amount of sodium tripolyphosphate aqueous solution to be added, the dropping is entered into the middle stage, the dropping speed is adjusted to 0.3% of the volume of the second mixture per minute, and the stirring speed is increased to 1350 r / min and maintained until the dropping is completed.

[0034] The aging process at 22°C for 3 hours described in step S4 is specifically carried out in a sealed container, which rotates horizontally around its central axis at a constant speed of 1.25 rpm.

[0035] It should be noted that the inventors discovered that when the above-mentioned 1.0% chitosan hydrochloride solution (pH approximately 5.2) and the β-cyclodextrin inclusion extract obtained in step S2 (pH approximately 5.5-6.0) are mixed at a ratio of 5:1, the resulting second mixture has a pH of approximately 5.3-5.8. This pH range falls precisely within the suitable pH range (4.0-6.0) for the cross-linking of chitosan and sodium tripolyphosphate. Although the subsequently added sodium tripolyphosphate solution (0.10%, pH approximately 9.2) is alkaline, because its addition volume is only 5% of the second mixture volume and a staged variable-rate addition method is used, local pH fluctuations are effectively controlled within the allowable range for cross-linking, and the overall pH of the system is maintained between 5.0 and 5.8, ensuring the smooth progress of the ionic cross-linking process.

[0036] Example 2 A method for preparing an antibacterial agent from mango peel extract (such as *Gynostemma pentaphyllum*) includes the following steps: S1. Dried mango peel powder, β-cyclodextrin, and deionized water were mixed to obtain a solid-liquid mixture. The mass-to-volume ratio of mango peel powder to deionized water was 1 g:17.5 mL, and the mass concentration of β-cyclodextrin relative to deionized water was 4.5% (i.e., 4.5 g of β-cyclodextrin was added per 100 mL or 100 g of deionized water). The solid-liquid mixture was placed in a high-voltage pulsed electric field treatment chamber and treated for 15 min under the conditions of an electric field strength of 25 kV / cm, a pulse width of 30 μs, and a pulse frequency of 150 Hz. During the treatment, the system temperature was maintained at 30 °C. After the treatment, the mixture was immediately transferred to a stirred reactor and stirred continuously for 4 h at 40 °C and a rotation speed of 400 r / min to obtain the extraction inclusion mixture. S2. Filter the extract-inclusion mixture obtained in step S1, collect the filtrate, and obtain a clear β-cyclodextrin inclusion extract; mix the β-cyclodextrin inclusion extract with chitosan hydrochloride solution at a volume ratio of 5:1 to obtain a second mixture; and shear the second mixture at 28°C and a speed of 12500 r / min for 3 min. S3. Under continuous stirring, sodium tripolyphosphate aqueous solution is added dropwise to the second mixture after high-speed shear dispersion. The mass concentration of the sodium tripolyphosphate aqueous solution is 0.10%, and the volume of the added solution is 5% of the volume of the second mixture. After the addition is completed, stirring is continued at 28°C for 60 min to obtain a nanoparticle suspension. S4. The nanoparticle suspension obtained in step S3 is mixed with a 60% (v / v) food-grade ethanol aqueous solution at a volume ratio of 12.5:1 and aged at 22℃ for 3 hours. After aging, the mixture is centrifuged at 10000 r / min for 20 minutes and the precipitate is collected. The precipitate is redispersed in a 0.01 mol / L citrate-sodium citrate buffer solution at a pH of 6.0 and a molar ratio of citric acid to sodium citrate of 1:2 at a ratio of 1 g to 25 mL. The solution is then filtered and sterilized using a 0.22 μm pore size filter membrane under positive pressure to obtain the antibacterial agent of Guiqi mango peel extract.

[0037] The chitosan hydrochloride solution is prepared by dissolving chitosan hydrochloride with a molecular weight range of 10-50 kDa and a degree of deacetylation greater than 90% in deionized water. The dissolution conditions are 25±5℃ and stirring at 500-800 r / min until completely dissolved, and the final solution mass concentration is 1.0%.

[0038] The dried mango peel powder is obtained by drying Guiqi mango peel with hot air (60~70℃) until the moisture content is ≤5.0%, and then pulverizing it to pass through a sieve with a pore size of 215μm.

[0039] The difference from Example 1 is the order in which the dried mango peel powder, β-cyclodextrin, and deionized water are mixed in step S1. Specifically, the process of mixing the dried mango peel powder, β-cyclodextrin, and deionized water in step S1 is as follows: First, all the prescribed amounts of β-cyclodextrin and deionized water are added and mixed at 30°C. Then, the mixture is sheared and dispersed at a high speed of 2500 r / min for 4 minutes to form a uniform pre-dispersed slurry. Subsequently, the prescribed amount of dried mango peel powder is added to the pre-dispersed slurry, and then the mixture is stirred and mixed at a speed of 500 r / min for 12.5 minutes to obtain the solid-liquid mixture system.

[0040] Unlike Example 1, in step S2, the extract-inclusion mixture obtained in step S1 is subjected to constant pressure filtration using a single-layer filter material. Specifically, a nylon microporous filter membrane with a pore size of 1.25 μm is used. During the filtration process, the vacuum pressure is controlled at -0.05 MPa and kept constant.

[0041] Unlike Example 1, step S3 does not use staged variable-speed dripping. Specifically, in step S3, when adding sodium tripolyphosphate aqueous solution dropwise to the second mixture under continuous stirring, the dripping rate is controlled to be 0.65% of the volume of the second mixture per minute, while the stirring speed is maintained at 1350 r / min until the dripping is completed.

[0042] Unlike Example 1, the aging process in step S4 is static aging, specifically: the aging process at 22°C for 3 hours is kept still in a sealed container.

[0043] Example 3 A method for preparing an antibacterial agent from Guiqi mango peel extract differs from Example 1 only in that, during the continuous stirring process described in step S1 for 4 hours, D-mannitol is added to the stirred reactor 2 hours after the start of stirring. The amount added is 20% of the mass of β-cyclodextrin in the solid-liquid mixture, and the remaining stirring time is continued. Everything else is exactly the same as in Example 1.

[0044] Example 4 A method for preparing an antibacterial agent from *Guiqi* mango peel extract differs from Example 1 only in that, after obtaining the extraction-inclusion mixture in step S1 and before filtration in step S2, an enzymatic hydrolysis and viscosity-reducing step is added: a compound enzyme preparation is added to the extraction-inclusion mixture for enzymatic hydrolysis; the compound enzyme preparation is composed of pectinase and cellulase in a mass ratio of 2.5:1, and its addition amount is 0.03% of the total mass of the extraction-inclusion mixture; the pectinase activity in the compound enzyme preparation is ≥50000 U / g, and the cellulase activity is ≥20000 U / g; the process conditions for enzymatic hydrolysis are: under continuous stirring, the pH of the system is controlled at 4.5 using dilute citric acid solution, the temperature is 40℃, and the hydrolysis time is 30 minutes; after the enzymatic hydrolysis is completed, the mixture is rapidly heated to 88℃ and maintained at this temperature for 6 minutes to completely inactivate the enzyme, then naturally cooled to 28℃ before proceeding to the filtration operation in step S2. Everything else is exactly the same as in Example 1.

[0045] It should be noted that the pH of the system was maintained at 4.5 during the enzymatic hydrolysis process, and remained around 4.5 after inactivation and cooling. The inventors discovered that this pH condition falls precisely within the suitable pH range (4.0-6.0) for chitosan-sodium tripolyphosphate crosslinking. When this enzymatic hydrolysate (pH 4.5) was mixed with chitosan hydrochloride solution (pH approximately 5.2), the system pH was approximately 5.0, still within the suitable crosslinking range. Subsequently, when adding sodium tripolyphosphate solution, because the amount of sodium tripolyphosphate (0.10%, pH approximately 9.2) added was only 5% of the volume of the second mixture, and a staged, variable-rate addition method was used, local pH fluctuations were effectively controlled, and the overall pH of the system was maintained between 5.0 and 5.8, ensuring the smooth progress of the ionic crosslinking process.

[0046] Comparative Example 1 The only difference between this comparative example and Example 2 is the order of operations in step S1, which employs a stepwise process of extraction followed by inclusion. Specifically, in step S1, the prescribed amount of dried mango peel powder and the prescribed amount of deionized water (mass-volume ratio 1g:17.5mL) are mixed and placed in a high-voltage pulsed electric field treatment chamber. The mixture is treated for 15 minutes under conditions of an electric field strength of 25kV / cm, a pulse width of 30μs, and a pulse frequency of 150Hz, while maintaining the system temperature at 30℃ during treatment. After treatment, the mixture is immediately transferred to a stirred reactor, and the prescribed amount of β-cyclodextrin is added to achieve a mass concentration of 4.5% relative to the deionized water (i.e., 4.5g of β-cyclodextrin per 100mL or 100g of deionized water). The mixture is stirred continuously for 4 hours at 40℃ and a rotation speed of 400r / min to obtain the extracted and included mixture. Subsequent steps S2 to S4 are identical to those in Example 2.

[0047] Comparative Example 2 The only difference between this comparative example and Example 2 is that the high-voltage pulse electric field treatment is not used in step S1.

[0048] Specifically, in step S1, dried mango peel powder, β-cyclodextrin, and deionized water are mixed according to the proportions in Example 2 to obtain a solid-liquid mixture. Without high-voltage pulsed electric field treatment, the mixture is directly transferred to a stirred reactor and stirred continuously for 4 hours at 40°C and 400 r / min to obtain the extraction and inclusion mixture. Subsequent steps S2 to S4 are exactly the same as in Example 2.

[0049] Comparative Example 3 The only difference between this comparative example and Example 2 is that chitosan hydrochloride solution is not added in step S2.

[0050] Specifically, in step S2, the extract-inclusion mixture obtained in step S1 is filtered, and the filtrate is collected to obtain a clear β-cyclodextrin inclusion extract. Without mixing with the chitosan hydrochloride solution, this β-cyclodextrin inclusion extract is directly sheared and dispersed at 28°C and a rotation speed of 12500 r / min for 3 min, and this is used as the "second mixture" in step S3. Steps S1, S3, and S4 are exactly the same as in Example 2.

[0051] Comparative Example 4 The only difference between this comparative example and Example 2 is that sodium tripolyphosphate aqueous solution is not added in step S3.

[0052] Specifically, in step S3, under continuous stirring, sodium tripolyphosphate aqueous solution is added to the second mixture after high-speed shear dispersion without adding dropwise, and stirring is continued at 28°C for 60 minutes to obtain a mixture (non-nanoparticle suspension). Steps S1, S2, and subsequent step S4 are exactly the same as in Example 2.

[0053] Comparative Example 5 The only difference between this comparative example and Example 2 is that ethanol aging and redispersion with citrate-sodium citrate buffer are not used in step S4.

[0054] Specifically, in step S4, the nanoparticle suspension obtained in step S3 is centrifuged directly at 10000 r / min for 20 min without mixing with the ethanol-water solution or aging, and the precipitate is collected. The precipitate is then redispersed directly in an equal volume of deionized water (precipitate mass: deionized water volume = 1 g: 25 mL) and filtered using a 0.22 μm pore size filter membrane to obtain the comparative product. Steps S1, S2, and S3 are exactly the same as in Example 2.

[0055] Comparative Example 6 The difference between this comparative example and Example 2 is that it uses the conventional ethanol solvent extraction method in the art instead of the high-voltage pulsed electric field-assisted β-cyclodextrin simultaneous extraction and inclusion process. Specifically: S1. Mix dried mango peel powder with 60% (v / v) food-grade ethanol aqueous solution at a mass-volume ratio of 1g:17.5mL to obtain an extraction system; extract the system in a 40℃ water bath at a speed of 400r / min for 4h; after extraction, centrifuge the mixture at 4000r / min for 15min, collect the supernatant, and remove the ethanol by rotary evaporation at 45℃ to obtain a concentrated aqueous extract.

[0056] S2. The concentrated aqueous extract obtained in step S1 is reconstituted with deionized water to the same volume as the β-cyclodextrin inclusion extract in Example 2, to obtain an extract. This extract is mixed with chitosan hydrochloride solution (mass concentration 1.0%) at a volume ratio of 5:1 to obtain a second mixture; the second mixture is then sheared and dispersed at 28°C and a rotation speed of 12500 r / min for 3 min.

[0057] S3. Under continuous stirring, add a 0.10% sodium tripolyphosphate aqueous solution dropwise to the second mixture after high-speed shear dispersion, with the added volume being 5% of the volume of the second mixture; after the addition is complete, continue stirring at 28℃ for 60 min to obtain a nanoparticle suspension.

[0058] S4. The nanoparticle suspension obtained in step S3 is mixed with a 60% (v / v) food-grade ethanol aqueous solution at a volume ratio of 12.5:1 and aged at 22°C for 3 hours. After aging, the mixture is centrifuged at 10000 r / min for 20 minutes and the precipitate is collected. The precipitate is redispersed in a 0.01 mol / L citrate-sodium citrate buffer solution at a ratio of 1 g:25 mL (precipitate mass: buffer volume) and filtered through a 0.22 μm filter membrane to remove bacteria, thus obtaining a contrast antibacterial agent.

[0059] Effect test: Experiment 1: Determination methods for active ingredient extraction rate and β-cyclodextrin encapsulation rate 1. Determination of total polyphenol extraction rate (Folin-phenol method) Principle: Polyphenols can reduce Folin-phenol reagent under alkaline conditions to form a blue complex, the color depth of which is directly proportional to the polyphenol content.

[0060] Reagents and instruments: Folin-phenol reagent, anhydrous sodium carbonate, gallic acid standard, 60% ethanol solution, analytical balance, ultrasonic instrument, constant temperature water bath, ultraviolet-visible spectrophotometer, centrifuge.

[0061] Preparation of standard curve: Accurately weigh gallic acid standard and prepare a series of standard solutions of different concentrations. Take different concentration standard solutions, add Folin-phenol reagent and sodium carbonate solution, and measure the absorbance at a wavelength of 760 nm to plot the standard curve (concentration-absorbance).

[0062] Sample determination: Sample pretreatment: Accurately weigh about 0.1 g of the lyophilized antibacterial agent powder finally prepared in each example / comparative example, dissolve it in 60% ethanol solution and make up to 25 mL, extract with ultrasonic assistance for 10 minutes, centrifuge and take the supernatant as the test solution.

[0063] Color development and determination: Take an appropriate amount of the test solution, carry out the color development reaction according to the standard curve preparation method, and measure its absorbance at 760 nm.

[0064] Calculation: Calculate the total polyphenol content (calculated as gallic acid, μg / mL) in the test solution according to the standard curve, and then calculate the total polyphenol extraction rate according to the sample mass and dilution factor.

[0065] Extraction rate (%) = (Total polyphenols in the sample / Mass of dried mango peel powder used) × 100% 2. Determination of encapsulation efficiency of specific active ingredients (ultrafiltration centrifugation-HPLC coupled method) Principle: Using ultrafiltration centrifuge tubes (molecular weight cutoff 10kDa), free, unencapsulated small molecule active ingredients are separated from nanoparticles. The contents of free components and total components are measured separately, and the encapsulation rate is calculated.

[0066] Reagents and instruments: methanol (chromatographic grade), phosphoric acid, acetonitrile (chromatographic grade), ultrafiltration centrifuge tubes (10kDa), high performance liquid chromatograph (equipped with C18 column and ultraviolet detector), high speed centrifuge, ultrasonic disruptor.

[0067] HPLC conditions: Chromatographic column: C18 reversed-phase column (4.6mm×250mm, 5μm).

[0068] Mobile phase: A (0.1% aqueous phosphoric acid solution), B (acetonitrile). Gradient elution program: 0-10 min, 15% B; 10-25 min, 15% → 45% B; 25-30 min, 45% → 15% B; 30-35 min, 15% B.

[0069] Flow rate: 1.0 mL / min.

[0070] Column temperature: 30℃.

[0071] Detection wavelengths: 258nm (mangiferin), 370nm (quercetin).

[0072] Injection volume: 10 μL.

[0073] Sample determination: Free component content (W) free Determination: Take 1.0 mL of each nanoparticle suspension sample, add it to an ultrafiltration centrifuge tube, and centrifuge at 12000 r / min and 4℃ for 30 minutes. Collect all filtrates and determine the concentrations of mangiferin and quercetin by HPLC.

[0074] Total component content (W) total Determination: Take another 1.0 mL of the same nanoparticle suspension sample, add 4.0 mL of methanol, vortex mix, and then sonicate (300 W power, 2 s operation, 3 s interval, 5 minutes in total) to completely destroy the nanoparticle structure. Then centrifuge at 12000 r / min for 10 minutes, take the supernatant, and determine the concentration of mangiferin and quercetin by HPLC.

[0075] Calculation: Encapsulation efficiency (%) = [(W total -W free ) / W total ×100%. The results are shown in Table 1.

[0076] Table 1: Extraction and encapsulation rates of active ingredients in the examples and comparative examples The results in Table 1 show that the integrated process of this invention (Example 1) achieves the highest total polyphenol extraction rate (8.2%) and excellent encapsulation rates for mangiferin and quercetin (92.5% and 88.7%, respectively), significantly better than Comparative Example 1, which uses a stepwise process (extraction rate 7.5%, encapsulation rates only 75.3% and 68.4%, respectively). This demonstrates that the synergistic process of high-voltage pulsed electric field and β-cyclodextrin achieves simultaneous extraction and encapsulation, offering advantages in improving efficiency and stabilizing active ingredients. Comparative Example 2, which omits the electric field treatment, shows a significant decrease in both extraction rate (4.1%) and encapsulation rate (approximately 61.2% and 52.8%), highlighting the crucial role of the electric field in cell disruption for the effective release of components. In terms of optimization, Example 3 further improved the encapsulation efficiency by adding D-mannitol (reaching 94.8% and 91.2%, respectively), demonstrating the effectiveness of this auxiliary step in enhancing inclusion. The encapsulation efficiency of Example 3 (with added D-mannitol) was also better than that of Example 1 (without added D-mannitol), indicating that adding D-mannitol at the 1.5-2.5h time point was indeed effective in "enhancing inclusion." In Example 4, the added enzymatic hydrolysis step resulted in indicators comparable to Example 1, showing that this step ensured process smoothness without compromising the extraction and inclusion effect. As a control, the traditional ethanol extraction method (Comparative Example 6) lacked inclusion effect because the extraction medium did not contain β-cyclodextrin. Furthermore, Comparative Examples 3 and 4, lacking chitosan or sodium tripolyphosphate respectively, could not construct complete nanoparticle carriers, and their encapsulation efficiency data were either meaningless or only showed extremely low physical adsorption rates, highlighting the necessity of a complete nanoparticle system for effectively encapsulating active ingredients.

[0077] Experiment 2: Detection Methods for Nanoparticle Size and Polydispersity Index Principle: Dynamic light scattering method. By measuring the fluctuations in the intensity of scattered light caused by the Brownian motion of nanoparticles in solution, the relationship between the fluctuation rate and particle size is analyzed, thereby obtaining the particle size distribution.

[0078] Instrument: Nanoparticle size and Zeta potential analyzer.

[0079] Sample preparation: Take the nanoparticle suspension products finally prepared in each example / comparative example and dilute them appropriately with their corresponding dispersion media (citric acid-sodium citrate buffer for comparative examples 1-5 and comparative examples 1-5, and the corresponding buffer for comparative example 6) until the sample solution is slightly opalescent or light blue and translucent, and the instrument count rate is within the optimal measurement range.

[0080] Measurement conditions: Temperature: 25.0±0.1℃.

[0081] Balancing time: 2 minutes.

[0082] Detection angle: 90°.

[0083] Each sample was measured three times.

[0084] Data Analysis: The instrument software directly outputs the average particle size (Z-average diameter, unit: nm) and polydispersity index (PDI). The closer the PDI value is to 0, the more uniform the particle size distribution; a PDI value greater than 0.3 is generally considered to indicate a wider distribution.

[0085] Auxiliary observation: Record the appearance of each sample after dilution (e.g., clarity, opalescence, turbidity, precipitation, etc.), and observe again after standing at room temperature for 24 hours to assess its physical stability. The results are shown in Table 2.

[0086] Table 2: Nanoparticle size and polydispersity index of examples and comparative examples Table 2 shows that Example 1, using the optimized process of this invention, achieved a smaller average particle size (182 nm) and a narrower particle size distribution (polydispersity index PDI of 0.18). Its suspension exhibited a uniform and stable appearance, demonstrating the synergistic effect of the optimized mixing, filtration, staged crosslinking, and rotary aging steps. This effectively controlled the formation and growth of nanoparticles, improving the uniformity of the formulation. In contrast, Example 2, which did not fully utilize the optimized steps, showed an increased particle size (205 nm) and a wider distribution (PDI 0.25), highlighting the contribution of each optimization step to improving nanoparticle properties. Based on Example 1, Examples 3 (with added D-mannitol) and 4 (with added enzymatic hydrolysis) maintained excellent particle size and PDI, indicating that these additional steps, while achieving their specific functions (enhanced inclusion and reduced viscosity), did not negatively impact the basic morphology of the nanoparticles. The particle size and PDI of Example 3 were even slightly better than those of Example 1, demonstrating that, from the perspective of final product quality, the addition of D-mannitol did not significantly negatively affect nanoparticle formation. In contrast, Comparative Example 2, lacking high-voltage pulsed electric field treatment, exhibited larger nanoparticle size (221 nm) and uneven distribution (PDI 0.28) due to insufficient extraction and complex system composition, leading to easy precipitation upon standing. Systems lacking key film-forming components (Comparative Examples 3 and 4) failed to form stable nano-dispersions, exhibiting rapid flocculation or severe aggregation, respectively. Furthermore, Comparative Example 5, without the ethanol aging step, showed a looser nanoparticle structure, with the largest particle size (255 nm) and the worst uniformity (PDI 0.31), highlighting the importance of this step for densifying the nanoparticle structure. Notably, Comparative Example 6, using traditional ethanol extraction followed by loading, also formed smaller (175 nm) and more uniform nanoparticles, but its overall performance (such as encapsulation efficiency and antibacterial activity) was inferior to the examples. This demonstrates that the advantage of this invention lies in the overall performance improvement brought about by the integrated process design from the source, rather than solely pursuing particle size parameters.

[0087] Experiment 3: Detection method for in vitro antibacterial activity (micro-broth dilution method) Principle: In a 96-well plate, the sample is serially diluted in liquid culture medium, and then inoculated with a certain concentration of test bacterial solution. After incubation, the lowest sample concentration at which no bacterial growth is observed by the naked eye is the minimum inhibitory concentration.

[0088] Strains and culture media: Test strains: Staphylococcus aureus (ATCC6538) and Escherichia coli (ATCC25922).

[0089] Culture medium: Mueller-Hinton (MH) broth medium.

[0090] Reagents and instruments: sterile 96-well cell culture plates, micropipette, colony counter, incubator, and clean bench.

[0091] Preparation of bacterial suspension: The activated test strain was inoculated into MH broth and cultured at 37°C with shaking until the logarithmic growth phase (approximately 18 hours). The bacterial suspension concentration was adjusted to 0.5 McFarland turbidity standard (approximately 1 × 10⁻⁶) with physiological saline. 8 (CFU / mL), then diluted 200 times with MH broth to obtain a final concentration of approximately 5 × 10⁻⁶ CFU / mL. 5 Working bacterial suspension at CFU / mL.

[0092] MIC determination procedure: Sample dilution: Add 200 μL of a high-concentration sample solution (e.g., 20 mg / mL) prepared in MH broth to the first column of wells in a 96-well plate. Starting from the first column, perform serial dilutions (i.e., pipette 100 μL into the next column of 100 μL MH broth, mix well, and repeat sequentially) until the 10th column. Column 11 contains no sample and serves as a growth control (positive control). Column 12 contains neither sample nor bacterial culture and serves as a sterility control (negative control).

[0093] Inoculation: Except for the negative control wells, 100 μL of working bacterial suspension was added to each of the other wells. At this point, the sample in each well was further diluted by 1-fold.

[0094] Culture: After covering the 96-well plate, place it in a 37℃ constant temperature incubator and incubate for 18-24 hours.

[0095] Results Interpretation: After incubation, the turbidity of each well was observed visually. The lowest sample concentration corresponding to a well that was completely clear with no visible bacterial growth was taken as the MIC value. Growth control wells should be significantly turbid, while sterile control wells should remain clear. The results are shown in Table 3, where the MIC values ​​are expressed in milligrams of lyophilized antimicrobial agent per milliliter (mg / mL).

[0096] Quality control: An antibiotic control (such as ampicillin) is set up for each test to ensure the sensitivity of the test strains and the effectiveness of the experimental system.

[0097] Table 3: Minimum In Vitro Inhibitory Concentrations of Examples and Comparative Examples Table 3 shows the in vitro antibacterial activity data, indicating that Example 1, prepared using the process of this invention, exhibited strong inhibitory effects against both Staphylococcus aureus and Escherichia coli, with minimum inhibitory concentrations (MICs) of 0.38 mg / mL and 1.25 mg / mL, respectively. This excellent activity is directly related to its efficient component extraction, high encapsulation efficiency, and uniform nanoparticle characteristics. In contrast, Comparative Example 1, using a stepwise process, and Comparative Example 6, using a traditional ethanol extraction method, showed significantly higher MIC values ​​(MICs against Staphylococcus aureus reached 0.65 mg / mL and 0.70 mg / mL, respectively), demonstrating the advantages of the integrated continuous process in maximizing the retention and delivery of active ingredients. When key steps were missing, the antibacterial activity decreased sharply: Comparative Example 2, without high-voltage pulsed electric field treatment, suffered the greatest activity loss (MIC value as high as 1.20 mg / mL), highlighting the necessity of efficient cell wall disruption extraction using an electric field; while Comparative Examples 3 and 4, which did not form complete nanoparticle carriers, also showed severely deteriorated activity, demonstrating the necessity of ion-crosslinked nanostructures for the protection and targeted delivery of active ingredients. Furthermore, in terms of optimization, Example 3, with the addition of D-mannitol, exhibited slightly better antibacterial activity than Example 1 (MIC of 0.35 mg / mL), which is consistent with the improved inclusion efficiency achieved by this step. In summary, it is evident that the integrated process of this invention, which achieves efficient extraction, stabilization, and nano-formulation of active ingredients, ultimately synergistically endows the final product with significantly enhanced antibacterial efficacy.

[0098] Experiment 4: Evaluation of sustained-release performance 1. Detection method: Dialysis bag method Principle: A suspension of nanoparticles is placed inside a dialysis bag (with a typical molecular weight cutoff of 8000-14000 Da), with a large amount of release medium outside the bag. The active ingredient is released from the nanoparticles and diffuses through the dialysis membrane into the external medium. The cumulative release of the active ingredient in the external medium is measured by taking samples at regular intervals, and a release curve is plotted to evaluate its release behavior.

[0099] Reagents and instruments: dialysis bags, phosphate buffer (PBS, pH 7.4), acetate-sodium acetate buffer (pH 5.0), constant temperature shaking water bath, high performance liquid chromatograph (HPLC).

[0100] Release conditions: To simulate different application environments (such as neutral body fluids or slightly acidic food spoilage microenvironments), two release media were set up: PBS (pH 7.4) and acetate-sodium acetate buffer (pH 5.0). The temperature was kept constant at 37±0.5℃.

[0101] Experimental steps: Sample preparation: Accurately measure the final nanoparticle suspension of each example / comparative example (equivalent to a certain amount of total polyphenols, such as 5 mg GAE), place it in a pretreated dialysis bag, and tie both ends tightly.

[0102] Release experiment: Immerse the dialysis bag in an Erlenmeyer flask containing 200 mL of the corresponding release medium and place it in a constant temperature shaking water bath (100 rpm).

[0103] Sampling and determination: At predetermined time points (e.g., 0.5, 1, 2, 4, 6, 8, 12, 24, 48 h), 2 mL of sample was taken from the external medium (while replenishing with an equal volume of fresh medium at the same temperature). The contents of mangiferin and quercetin in the taken sample were determined by HPLC.

[0104] Calculation: Based on the measured concentrations, calculate the cumulative release rate of the two active ingredients at each time point.

[0105] Cumulative release rate (%) = (Total amount of active ingredient released up to the current time point / Initial total amount of that active ingredient in the nanoparticles) × 100% Data analysis: Plot a "time-cumulative release rate" curve. Evaluate the sustained-release performance of different samples by comparing their release rates at the same time point, the time to reach the release plateau (e.g., 80% release), and the shape of the release curve (whether it is flat or has a burst release).

[0106] 2. Experimental Data To provide a clearer picture, the cumulative release rate at the key 24-hour time point was selected for comparison. The experimental results are shown in Table 4.

[0107] Table 4: Cumulative in vitro release rate of active ingredients at 24 hours in the Examples and Comparative Examples Note: In Comparative Example 6, quercetin, not being encapsulated by β-cyclodextrin, may rapidly dissolve or undergo physical precipitation in release media containing organic phases or surfactants. Its "release" behavior does not conform to the nanoparticle controlled-release model. *Data characterizes its rapid dissolution from the formulation. "Rapid release (>90% in 2h)" means rapid release of more than 90% within 2 hours.

[0108] The in vitro release data in Table 4 show that the antibacterial agent prepared in this invention exhibits significant sustained-release characteristics and pH responsiveness. Example 1 showed the most gradual cumulative release rate over 24 hours, indicating its most dense and stable nanoparticle structure. In contrast, Comparative Examples 3 and 4, lacking key structural components, showed rapid and complete release of their active ingredients within a very short time, without any sustained-release effect. This demonstrates that intact ion-crosslinked nanoparticles are essential carriers for controlled release. Furthermore, all granulated samples exhibited faster release rates in a simulated acidic environment, indicating the unique pH-responsive release behavior of their chitosan-based nanoparticles, suggesting potentially faster efficacy in acidic application scenarios. Therefore, the composite nanoparticles constructed using the integrated process of this invention can achieve long-term, controlled release of the active ingredients.

[0109] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A method for preparing an antibacterial agent from mango peel extract, characterized in that, Includes the following steps: S1. Mix dried mango peel powder, β-cyclodextrin, and deionized water to obtain a solid-liquid mixture system; wherein the mass-to-volume ratio of mango peel powder to deionized water is 1g:15-20mL, and the mass concentration of β-cyclodextrin relative to deionized water is 3%-6%; place the solid-liquid mixture system under an electric field strength of 20-30kV / cm, a pulse width of 20-40μs, and a pulse frequency of 100-200Hz for 10-20min, and maintain the system temperature at 25-35℃ during the treatment; after the treatment, immediately transfer the mixture system to a stirred reactor and stir continuously at 35-45℃ and a rotation speed of 300-500r / min for 3-5h to obtain an extraction inclusion mixture; S2. Filter the extract-inclusion mixture obtained in step S1, collect the filtrate, and obtain a clear β-cyclodextrin inclusion extract; mix the β-cyclodextrin inclusion extract with chitosan hydrochloride solution at a volume ratio of 4-6:1 to obtain a second mixture; disperse the second mixture at 25-30℃ and a high-speed shearing speed of 10000-15000 r / min for 2-4 min. S3. Under continuous stirring at 800-1500 r / min, sodium tripolyphosphate aqueous solution is added dropwise to the second mixture after high-speed shear dispersion. The mass concentration of the sodium tripolyphosphate aqueous solution is 0.08-0.12%, and the volume of the added solution is 4-6% of the volume of the second mixture. After the addition is complete, stirring is continued at 25-30℃ for 40-80 min to obtain a nanoparticle suspension. S4. The nanoparticle suspension obtained in step S3 is mixed with a 50-70% (v / v) food-grade ethanol aqueous solution at a volume ratio of 10-15:1 and aged at 20-25℃ for 2-4 hours. After aging, the mixture is centrifuged at 8000-12000 r / min for 15-25 min. The wet precipitate collected by centrifugation is redispersed in a citrate-sodium citrate buffer solution with a pH of 5.5-6.5 and a concentration of 0.01 mol / L at a ratio of 1 g of precipitate to 20-30 mL of buffer solution. The solution is then filtered through a 0.22 μm filter membrane for sterilization to obtain the antibacterial agent of Guiqi mango peel extract.

2. The preparation method of the antibacterial agent from the peel extract of *Gynostemma pentaphyllum* as described in claim 1, characterized in that, The chitosan hydrochloride solution is prepared by dissolving chitosan hydrochloride with a degree of deacetylation greater than 90% in deionized water, resulting in a chitosan hydrochloride solution with a mass concentration of 0.8-1.2%.

3. The preparation method of the antibacterial agent from the peel extract of *Gynostemma pentaphyllum* as described in claim 1, characterized in that, The dried mango peel powder is obtained by pulverizing dried Guiqi mango peel and passing it through a sieve with a pore size of 180-250μm.

4. The preparation method of the antibacterial agent from the peel extract of *Gynostemma pentaphyllum* as described in claim 1, characterized in that, The specific process of mixing dried mango peel powder, β-cyclodextrin, and deionized water in step S1 is as follows: First, the β-cyclodextrin of the formula amount is mixed with deionized water accounting for 60-70% of the total water required by the formula at 25-35°C, and then sheared and dispersed at a high speed of 2000-3000 r / min for 3-5 minutes to form a uniform pre-dispersed slurry; then, the dried mango peel powder of the formula amount is added to the pre-dispersed slurry, and the remaining deionized water is added to the total water required by the mass-volume ratio, and then stirred and mixed at a speed of 400-600 r / min for 10-15 minutes to obtain the solid-liquid mixture system.

5. The preparation method of the antibacterial agent from the peel extract of *Gynostemma pentaphyllum* as described in claim 1, characterized in that, The specific process of filtration of the extract and inclusion mixture obtained in step S1 in step S2 is as follows: constant pressure filtration is performed using a double-layer filter material, which consists of an upper layer of qualitative filter paper and a lower layer of nylon microporous filter membrane. The pore size of the qualitative filter paper is 10-15 μm, and the pore size of the nylon microporous filter membrane is 1.0-1.5 μm. During the filtration process, the vacuum pressure is controlled within the range of -0.04 MPa to -0.06 MPa and kept constant.

6. The preparation method of the antibacterial agent from the peel extract of *Gynostemma pentaphyllum* as described in claim 1, characterized in that, The aging process described in step S4 at 20-25°C for 2-4 hours is specifically carried out in a sealed container, which rotates horizontally around its central axis at a speed of 0.5-2 rpm continuously and at a constant speed.

7. The method for preparing the antibacterial agent from the peel extract of *Gynostemma pentaphyllum* as described in claim 1, characterized in that, In step S3, when adding sodium tripolyphosphate aqueous solution dropwise to the second mixture under continuous stirring, a staged variable-speed dropping method is adopted, which is coordinated with the corresponding stirring speed: in the initial stage of dropping, the dropping speed is controlled at 0.5-0.8% of the volume of the second mixture per minute, while the stirring speed is maintained at 800-1000 r / min; when the dropping volume reaches 40%-50% of the total amount of sodium tripolyphosphate aqueous solution to be added, the dropping is entered into the middle stage, and the dropping speed is adjusted to 0.2%-0.4% of the volume of the second mixture per minute, while the stirring speed is increased to 1200-1500 r / min and maintained until the dropping is completed.

8. The method for preparing the antibacterial agent of *Gynostemma pentaphyllum* mango peel extract as described in any one of claims 1-7, characterized in that, During the continuous stirring process described in step S1 for 3-5 hours, 1.5-2.5 hours after the start of stirring, D-mannitol is added to the stirred reactor. The amount added is 15%-25% of the mass of β-cyclodextrin in the solid-liquid mixture, and the remaining stirring time is continued.

9. The method for preparing the antibacterial agent of *Gynostemma pentaphyllum* mango peel extract as described in any one of claims 1-7, characterized in that, After obtaining the extraction and inclusion mixture in step S1 and before filtration in step S2, an enzymatic hydrolysis and viscosity reduction step is added: a compound enzyme preparation is added to the extraction and inclusion mixture for enzymatic hydrolysis; the compound enzyme preparation is composed of pectinase and cellulase in a mass ratio of 2-3:1, and its addition amount is 0.02-0.05% of the total mass of the extraction and inclusion mixture; the pectinase activity in the compound enzyme preparation is ≥50000U / g, and the cellulase activity is ≥20000U / g; the process conditions for enzymatic hydrolysis are: under continuous stirring, the system temperature is controlled at 38-42℃, the pH value is 4.0-5.0, and the hydrolysis time is 20-40 minutes; after the enzymatic hydrolysis is completed, the mixture is rapidly heated to 85-90℃ and maintained at this temperature for 5-10 minutes to completely inactivate the enzyme, and then naturally cooled to 25-30℃ before proceeding to the filtration operation in step S2.

10. A bactericidal agent derived from the peel extract of *Gynostemma pentaphyllum* mango, characterized in that, It is prepared by the method for preparing the antibacterial agent of Guiqi mango peel extract according to any one of claims 1-7.

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