A method for inhibiting lignification of loquat fruit due to postharvest chilling injury.

Abstract

This invention discloses a method for inhibiting postharvest chilling injury and lignification of loquat fruit, comprising the following steps: (1) preparation of MeJA@NPs nanoparticle solution; (2) preparation of GP-MeJA@NPs coating solution; (3) in-situ coating processing. The materials used in this invention have high biocompatibility, do not cause environmental pollution, have high raw material utilization, require only low concentrations to produce good treatment effects, have good sustained treatment effects, are simple to operate, and have low cost, making them suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of agricultural product preservation technology, and in particular to a method for inhibiting postharvest chilling injury and lignification of loquat fruit. Background Technology

[0002] Loquat (Eriobotrya japonica Lindl.) belongs to the genus Eriobotrya in the family Rosaceae. It is a typical subtropical evergreen small tree, with most loquats naturally distributed between 20° and 35° north latitude. Loquat originated in China and has a cultivation history of nearly 2000 years. Currently, my country is the world's largest loquat producer, accounting for over 80% of global production, with major production areas concentrated in Zhejiang, Jiangsu, Sichuan, Chongqing, and Fujian provinces. Loquat fruits can be divided into two categories based on the color of the flesh: red-fleshed and white-fleshed. In Zhejiang, common red-fleshed loquat varieties include 'Da Hong Pao', 'Luoyang Qing', and 'Dan Bian Zhong', while common white-fleshed varieties include 'Ruan Tiao Bai Sha', 'Ninghai Bai', and 'Bai Jing No. 1'. Loquat is one of the specialty fruits of Zhejiang Province, with a planting area exceeding 1.5 hectares in 2019. 2 The output exceeded 100,000 tons, with a value of approximately 1.65 billion yuan.

[0003] Ripe loquats are soft, juicy, and sweet-and-sour, rich in nutrients, including carbohydrates, organic acids, vitamins, and various essential trace elements such as calcium, magnesium, iron, manganese, and zinc, while being low in calories and fat. Loquat fruit also has cough-relieving, lung-moistening, and digestive properties, while loquat leaves have anti-inflammatory, antioxidant, and blood sugar-lowering effects. Therefore, loquats are very popular with consumers.

[0004] Loquat fruits typically ripen and are harvested during the hot and rainy early summer. After harvesting, the fruit is not suitable for storage at room temperature and rots very easily. Low-temperature storage is a common strategy for extending the shelf life of loquat fruits and can effectively inhibit rotting, but it can also easily lead to chilling injury and lignification. Chilling injury and lignification in loquat fruits manifest as increased lignin content and increased fruit firmness, accompanied by decreased juice yield, browning of the flesh, and difficulty in peeling the skin, severely affecting the flavor and quality of the fruit.

[0005] Low temperature conditioning (LTC) is a commonly used method for treating chilling injury in fruits. It involves pre-storing the fruits in an environment slightly above the critical chilling injury temperature to reduce chilling injury during subsequent low-temperature storage.

[0006] Heat treatment, also known as heat shock treatment, generally involves treating fruits with hot water, hot air, hot steam, far-infrared rays, or microwaves (35-50°C) to alter fruit enzyme activity, induce enhanced fruit resistance, and thus reduce chilling injury symptoms during subsequent low-temperature storage. Rui Huaijin found that during storage at 1°C, hot air treatment (38°C, 5h) on 'Jiefangzhong' loquat fruits could slow down chilling injury lignification, inhibit the activity of phenylalanine ammonia-lyase (PAL), peroxidase (POD), and polyphenol oxidase (PPO), reduce the increase in lignin content, while maintaining high activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), and reducing the accumulation of O2- and malondialdehyde (MDA) (Study on the Effects and Mechanisms of CaCl2 and Heat Treatment on Lignification of Loquat Fruits).

[0007] Controlled atmosphere storage mainly relies on altering the concentrations of CO2 and O2 in the fruit storage environment to control post-harvest ripening and senescence. Xiang Shijie effectively controlled the accumulation of lignin in 'Da Wuxing' loquat fruit during storage at 5℃ using controlled atmosphere storage (5% O2 + 5% CO2), and delayed the increase in PAL and POD enzyme activities (Application Study of Controlled Atmosphere Storage and 1-MCP Preservation Treatment on 'Da Wuxing' Loquat Fruit).

[0008] Methyl jasmonic acid (MeJA) is the methyl esterified form of jasmonic acid and a volatile compound widely found in higher plants. Exogenous MeJA can enter plants through stomata, inducing defense responses and enabling long-distance signal transduction and communication. When plants are subjected to stresses such as drought, low temperature, high salinity, heavy metals, and pests and diseases, exogenous MeJA can act as a signaling molecule to induce the expression of related resistance genes or maintain the activity of related enzymes, thereby enhancing plant stress resistance. MeJA also has other broad physiological effects, such as promoting the production of plant secondary metabolites, regulating plant photosynthesis, and regulating the timing and quantity of flowering. Due to its non-toxic, harmless, environmentally friendly, and broad-spectrum characteristics, many researchers have applied MeJA to the study of postharvest chilling injury inhibition in fruits. In loquat research, Cao Shifeng discovered that 10 μmol L... -1 MeJA fumigation treatment (24h, 20℃) can significantly inhibit the activities of PAL, 4CL, PPO, cinnamyl alcohol dehydrogenase (CAD), and POD in 'Fuyang' loquat fruits during storage at 1℃, reducing lignin content and reactive oxygen species accumulation in the fruits, thereby alleviating chilling injury symptoms in loquats (Research on the regulation and mechanism of postharvest quality deterioration of loquat fruits). However, fumigation still has problems such as low treatment efficiency, difficulty in controlling concentration, waste of treatment materials, easy generation of residues, high long-term treatment costs, and poor short-term treatment effect sustainability.

[0009] Active packaging is a form of packaging loaded with functional active ingredients. It primarily achieves specific functions by releasing or absorbing substances from the packaged object or its surrounding environment through the loaded active ingredients. Given the prevalence of post-harvest fruit quality deterioration, spoilage, and mechanical damage, active packaging with functions such as preservation, antibacterial properties, and antioxidants is currently a research hotspot in post-harvest fruit storage and preservation. Summary of the Invention

[0010] The purpose of this invention is to provide a method for inhibiting postharvest chilling injury and lignification of loquat fruit. The materials used have high biosafety, do not cause environmental pollution, have high raw material utilization, require only low concentration to produce good treatment effect, have good treatment effect, are simple to operate, have low cost, and are suitable for industrial production.

[0011] The technical solution adopted by this invention to solve its technical problem is: A method for inhibiting lignification of loquat fruit due to postharvest chilling injury includes the following steps: (1) Preparation of MeJA@NPs nanoparticle solution: MeJA was added to a carboxymethyl chitosan solution to form a mixed solution of carboxymethyl chitosan and MeJA. Under stirring conditions, CaCl2 solution was added dropwise to the mixed solution of carboxymethyl chitosan and MeJA at a uniform rate and mixed well to form a carboxymethyl chitosan nanoparticle solution loaded with MeJA, namely MeJA@NPs nanoparticle solution. (2) Preparation of GP-MeJA@NPs coating solution: Gelatin and pullulan were dissolved in water to form a mixed solution. Then, glycerol was added and stirred until homogeneous to form a GP coating solution. The GP coating solution was mixed with an equal volume of MeJA@NPs nanoparticle solution to obtain a GP-MeJA@NPs coating solution. (3) In-situ coating processing: Soak loquat fruits in GP-MeJA@NPs coating solution at room temperature for 10-15 minutes, then remove and air dry.

[0012] In step (1), the volume ratio of CaCl2 solution to the mixed solution of carboxymethyl chitosan and MeJA is 1:2-3. Controlling the ratio of calcium chloride to carboxymethyl chitosan is key to forming stable nanoparticles.

[0013] Preferably, the volume ratio of CaCl2 solution to the mixed solution of carboxymethyl chitosan and MeJA is 1:2.5. A volume ratio of 1:2.5 results in smaller particle diameters and more uniform particle size.

[0014] In step (1), the mixed solution of carboxymethyl chitosan and MeJA has the following concentrations: carboxymethyl chitosan concentration is 0.01-0.02 g / mL and MeJA concentration is 25-32 μmol / L.

[0015] Preferably, in the mixed solution of carboxymethyl chitosan and MeJA, the concentration of carboxymethyl chitosan is 0.01 g / mL and the concentration of MeJA is 28 μmol / L. At room temperature, the solubility of methyl jasmonate in water is 1500 μmol / L.

[0016] In step (1), the CaCl2 solution is added at a rate of 3-5 mL / min.

[0017] In step (2), the volume concentration of glycerol in the GP coating solution is 0.5-1%, and the total concentration of gelatin and pullulan is 0.01-0.02 g / mL.

[0018] Preferably, the mass ratio of gelatin to pullulan is 1-2:1.

[0019] Gelatin (GL), pullulan (PUL), and chitosan, as common biopolymers, possess excellent biodegradability, non-toxicity, and film-forming properties. Gelatin-based films and edible coatings serve as carriers for the controlled release of bioactive molecules; pullulan improves the thermal stability of films and reduces moisture evaporation; chitosan is a good film-forming material, and chitosan coatings have been shown to reduce loquat fruit decay. Therefore, GL, PUL, and chitosan are ideal substrate materials for fruit preservation films, serving as controlled-release carriers for MeJA (meta-alcoholic acid), and as bio-based packaging, they can reduce the environmental pollution problems caused by traditional packaging such as plastics. Carboxymethyl chitosan (CMCS), a water-soluble chitosan derivative, is used in this invention to replace acid-soluble chitosan as a substrate material for bio-based active packaging to address the issue of trace acetic acid residue.

[0020] The beneficial effects of this invention are: 1. Using gelatin, pullulan, carboxymethyl chitosan and MeJA as raw materials, loquat is encapsulated in situ, which has high biosafety and does not cause environmental pollution.

[0021] 2. Carboxymethyl chitosan nanoparticles loaded with MeJA are formed by ion crosslinking, which has high encapsulation efficiency for MeJA and good sustained-release effect.

[0022] 3. It is simple to operate and has low cost; the cost of processing a single loquat is about 0.05 yuan. Attached Figure Description

[0023] Figure 1 These are rheological test results for the coating solution: (a) viscosity changes with shear rate; (b) in-situ coating treatment diagram. Figure 2 These are scanning electron microscope images of nanoparticles, (a) NPs and (b) MeJA@NPs; Figure 3 These are scanning electron microscope images of the coated films: (a) GP coated film, (b) GP-NPs coated film and (c) GP-MeJA@NPs coated film; Figure 4 These are Fourier transform infrared (FTIR) spectra of nanoparticles and coating films, (a) nanoparticles, (b) coating; Figure 5 This is a thermogravimetric analysis (TGA) curve of nanoparticles and coating films; Figure 6 These are the MeJA release amounts and fitted curves of MeJA@NPs; Figure 7 Fluorescence micrographs of GES-1 cell viability: (a) blank control and (b) GP-MeJA@NPs; Figure 8 The graph shows the hardness and lignin content of loquat fruits in each treatment group: (a) hardness, (b) lignin content. Figure 9 The following is a graph showing the activity of enzymes related to phenylpropane metabolism in loquat fruits from each treatment group: (a) phenylalanine ammonia-lyase (PAL); (b) cinnamic acid-4-hydroxylase (C4H); (c) 4-coumaric acid-coenzyme A ligase (4CL); (d) peroxidase (POD); different lowercase letters indicate significant differences between groups at that time point; Figure 10 The graph shows the changes in total phenolic and total flavonoid content in loquat fruits of each treatment group: (a) total phenolic content, (b) total flavonoid content; Figure 11 The following is a graph showing the activity of enzymes related to oxidative metabolism in loquat fruit: (a) superoxide dismutase (SOD); (b) catalase (CAT); (c) ascorbate peroxidase (APX). Different lowercase letters indicate significant differences between groups at that time point. Figure 12 The graph shows the changes in oxidative metabolites of loquat fruit in each treatment group: (a) hydrogen peroxide (H2O2), (b) malondialdehyde (MDA) content. Figure 13 This is a graph showing the changes in proline (Pro) content in loquat fruits from different treatment groups. Detailed Implementation

[0024] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0025] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. The methods described in the following embodiments are conventional methods in the field, unless otherwise specified.

[0026] The inventors previously prepared a slow-release composite film using gelatin, pullulan, chitosan, and MeJA. Although this film could inhibit the lignification of loquat fruit due to postharvest chilling injury, it had problems such as high cost, low encapsulation efficiency, and the potential for trace amounts of acetic acid residue.

[0027] Carboxymethyl chitosan (degree of deacetylation ≥90%, Hefei Bomei Biotechnology Co., Ltd.), calcium chloride (CaCl2, 110.98MW, Coolaber), gelatin (gel strength 250g, Aladdin), pullulan (Macklin), glycerol (99%, 92.09MW, Macklin), methyl jasmonate (MeJA, 224.3MW, 95%, Macklin), thiazolyl blue (MTT, 98%, Macklin), phosphate buffered saline (PBS, 10×, Solarbio), dimethyl sulfoxide (99.7%, Macklin).

[0028] Example 1: A method for inhibiting lignification of loquat fruit due to postharvest chilling injury includes the following steps: (1) Preparation of MeJA@NPs nanoparticle solution: Under stirring at 1000 rpm, 0.01 g / mL CaCl2 solution was added dropwise at a constant rate of 3 mL / min to a mixed solution of carboxymethyl chitosan and MeJA and mixed well to form a MeJA-loaded carboxymethyl chitosan nanoparticle solution, i.e., MeJA@NPs nanoparticle solution; the volume ratio of CaCl2 solution to mixed solution of carboxymethyl chitosan and MeJA was 1:2; the concentration of carboxymethyl chitosan in the mixed solution of carboxymethyl chitosan and MeJA was 0.01 g / mL and the concentration of MeJA was 25 μmol / L.

[0029] (2) Preparation of GP-MeJA@NPs coating solution: Gelatin and pullulan were dissolved in water to form a mixed solution. Then, glycerol was added and stirred until homogeneous to form a GP coating solution. In the GP coating solution, the volume concentration of glycerol was 0.5%, the total concentration of gelatin and pullulan was 0.01 g / mL, and the mass ratio of gelatin to pullulan was 1:1. The GP coating solution was mixed with an equal volume of MeJA@NPs nanoparticle solution to obtain the GP-MeJA@NPs coating solution.

[0030] (3) In-situ coating processing: Soak loquat fruits in GP-MeJA@NPs coating solution at room temperature for 10 minutes, then remove and air dry.

[0031] Example 2: A method for inhibiting lignification of loquat fruit due to postharvest chilling injury includes the following steps: (1) Preparation of MeJA@NPs nanoparticle solution: Under stirring at 1000 rpm, 0.01 g / mL CaCl2 solution was added dropwise at a constant rate of 5 mL / min to a mixed solution of carboxymethyl chitosan and MeJA to form a MeJA-loaded carboxymethyl chitosan nanoparticle solution, i.e., MeJA@NPs nanoparticle solution; the volume ratio of CaCl2 solution to mixed solution of carboxymethyl chitosan and MeJA was 1:3; the concentration of carboxymethyl chitosan in the mixed solution of carboxymethyl chitosan and MeJA was 0.02 g / mL, and the concentration of MeJA was 32 μmol / L.

[0032] (2) Preparation of GP-MeJA@NPs coating solution: Gelatin and pullulan were dissolved in water to form a mixed solution. Then, glycerol was added and stirred evenly to form a GP coating solution. In the GP coating solution, the volume concentration of glycerol was 1%, the total concentration of gelatin and pullulan was 0.02 g / mL, and the mass ratio of gelatin to pullulan was 2:1. The GP coating solution was mixed with an equal volume of MeJA@NPs nanoparticle solution to obtain the GP-MeJA@NPs coating solution.

[0033] (3) In-situ coating processing: Soak loquat fruits in GP-MeJA@NPs coating solution at room temperature for 15 minutes, then remove and air dry.

[0034] Example 3: A method for inhibiting lignification of loquat fruit due to postharvest chilling injury includes the following steps: (1) Preparation of MeJA@NPs nanoparticle solution: Under stirring at 1000 rpm, 0.01 g / mL CaCl2 solution was added dropwise at a constant rate of 4 mL / min to a mixed solution of carboxymethyl chitosan and MeJA to form a MeJA-loaded carboxymethyl chitosan nanoparticle solution, i.e., MeJA@NPs nanoparticle solution; the volume ratio of CaCl2 solution to mixed solution of carboxymethyl chitosan and MeJA was 1:2.5; the concentration of carboxymethyl chitosan in the mixed solution of carboxymethyl chitosan and MeJA was 0.01 g / mL, and the concentration of MeJA was 28 μmol / L.

[0035] (2) Preparation of GP-MeJA@NPs coating solution: Gelatin and pullulan were dissolved in water to form a mixed solution. Then, glycerol was added and stirred evenly to form a GP coating solution. In the GP coating solution, the volume concentration of glycerol was 0.5%, the total concentration of gelatin and pullulan was 0.01 g / mL, and the mass ratio of gelatin to pullulan was 1:1. The GP coating solution was mixed with an equal volume of MeJA@NPs nanoparticle solution to obtain the GP-MeJA@NPs coating solution.

[0036] (3) In-situ coating processing: Soak loquat fruits in GP-MeJA@NPs coating solution at room temperature for 10 minutes, then remove and air dry.

[0037] Experimental preparation example: 4 g of CMCS (carboxymethyl chitosan) was added to 400 mL of purified water and stirred at 50 °C to dissolve, thus preparing a 1% CMCS solution (w / v). After cooling, the magnetic stirrer was set to 1000 rpm, and 160 mL of 1% CaCl2 solution was added at 4 mL / min using a constant pressure dropping funnel. -1 The solution was added dropwise at a constant rate to the CMCS solution (CaCl2 solution:CMCS solution volume ratio = 2:5) to obtain a CMCS nanoparticle solution, which was named NPs solution.

[0038] The preparation process of the MeJA-loaded nanoparticle solution is as follows: MeJA is added to a 0.01 g / mL CMCS solution until the concentration reaches 28 μmol / L. -1 The mixture was stirred continuously at 25°C for 15 minutes. Then, with stirring at 1000 rpm, 160 mL of 1% CaCl2 solution was added dropwise at a constant pressure dropping funnel to 400 mL of a mixed solution of CMCS and MeJA, ultimately obtaining a MeJA-loaded CMCS nanoparticle solution (MeJA concentration approximately 20 μmol / L). -1 ), and named it MeJA@NPs solution.

[0039] Nanoparticles uniformly dispersed in a solution system were collected using a freeze-drying method. The preparation process was as follows: NPs solution and MeJA@NPs solution were centrifuged at 8000 rpm for 30 min, the supernatant was removed, and the precipitate was washed twice with purified water. The precipitate was then ultrasonically dispersed in purified water and stored overnight at -20℃ to allow the solution to freeze. Finally, it was dried in a freeze dryer (LGJ-10C10, Sihuan Furui Instrument Technology Development Co., Ltd.) for 2 days to obtain freeze-dried NPs and MeJA@NPs.

[0040] Preparation of coating solution 2.8 g each of gelatin (GL) and pullulan (PUL) were added to 560 mL of purified water and stirred at 50 °C to dissolve, preparing a 1% (w / v) polymer solution. Then, 2.8 mL of glycerol was added as a plasticizer (0.5% v / v based on the polymer solution volume), and the mixture was stirred continuously for 30 min to obtain a GL / PUL mixed solution, which was named the GP coating solution. The nanoparticle coating solution was then prepared using a blending method: the GP coating solution was mixed with an NPs solution at a 1:1 ratio (v / v), and stirred to obtain a homogeneous and stable GP-NPs coating solution. Similarly, the GP coating solution was mixed with a MeJA@NPs solution at a 1:1 ratio (v / v), and stirred to obtain a homogeneous and stable GP-MeJA@NPs coating solution. The MeJA concentration in the MeJA nanoparticle coating solution was approximately 10 μmol / L. -1 .

[0041] Preparation of nanoparticle coated thin films Coated films were prepared using a solution casting method with GP coating solution, GP-NPs coating solution, and GP-MeJA@NPs coating solution: 120 mL of film-forming solutions with different proportions were added to 25 cm × 25 cm polystyrene square petri dishes and dried in an oven at 65 °C for 6 h, during which time the petri dishes were kept horizontal. The dried composite films were peeled off and stored for subsequent experiments.

[0042] Rheological properties determination of nanoparticle coated solution The rheological properties of the coating solution were determined using a rheometer (Haake Mars 40, Germany). The instrument had a cone diameter of 60 mm and a shear rate range of 0.1 s⁻¹. -1 ~1000s -1 The test mode was kinetic viscosity test (rotation mode), and the test was repeated 3 times.

[0043] In-situ coating effects were tested on 'Luoyangqing' loquat fruit (Eriobotrya japonica Lindl.cv.Luoyangqing). Fruit samples were sourced from orchards in Taizhou City, Zhejiang Province. The testing procedure was as follows: Group 1: Loquat fruit without any treatment served as a control group; Group 2: Loquat fruit samples were immersed in GP-NPs coating solution for 10 minutes and then air-dried; Group 3: Loquat fruit samples were immersed in GP-MeJA@NPs coating solution for 10 minutes and then air-dried. Finally, the in-situ coating effect on the fruit was photographed and the coating film effect was tested.

[0044] like Figure 1As shown in Figure a, in the rheological test, the curve trends of the GP-NPs and GP-MeJA@NPs coating solutions almost overlapped, exhibiting nonlinear changes similar to the GP solution, indicating shear thinning, thus demonstrating that the coating solutions are non-Newtonian fluids. The initial viscosities of the GP, GP-NPs, and GP-MeJA@NPs coating solutions were 15.6 mPa·s, 14.1 mPa·s, and 19.1 mPa·s, respectively, all belonging to low viscosity. Low-viscosity coating solutions can form thinner and more uniform coating films on the fruit surface. Figure 1 As shown in b, the GP-MeJA@NPs coating solution can form a thin and uniform coating film on the surface of loquat fruit, with no significant difference compared to the control group, indicating that the coating treatment does not affect the appearance of the fruit or subsequent sales. Furthermore, the GP-MeJA@NPs coating can protect the surface of the loquat fruit from scratches.

[0045] Observation of surface morphology of nanoparticles First, the GP-NPs coating solution and the GP-MeJA@NPs coating solution were contacted with distilled water through an ion exchange membrane for one day to remove excess residual salt. Then, a certain amount of the nanoparticle coating solution was diluted 100 times with pure water. Finally, a small amount of the diluted nanoparticle solution was dropped onto a silicon wafer, dried, and then subjected to gold sputtering. The surface microstructure was observed using a scanning electron microscope (EISS Sigma 300, Germany).

[0046] SEM results are as follows Figure 2 As shown, most NPs are approximately spherical particles, with a small portion appearing as elongated strips. MeJA@NPs, however, do not appear as single particles, but rather as clusters of many small particles aggregated together, exhibiting a clump-like structure, with the diameter of these clump-like particles significantly larger than that of the NPs. These results demonstrate that MeJA@NPs successfully encapsulate MeJA.

[0047] Observation of the surface morphology of the coating film All coated film samples underwent gold sputtering treatment, and their surface microstructure was then observed using a scanning electron microscope (Hitachi SU8010, Japan). The SEM results of the coated films are shown below. Figure 3 As shown, the matrix of the GP coating film is relatively uniform. Figure 3 a) The surface is continuous, indicating that GL and PUL are well mixed. After the addition of nanoparticles, GP-NPs ( Figure 3 b) and GP-MeJA@NPs( Figure 3 c) The surface of the coating film becomes rough and uneven, possibly because the addition of nanoparticles disrupts the original uniform matrix structure of the film. These results indicate that nanoparticles can be fully mixed with GP to form a film and embedded within the coating film, ultimately achieving adhesion to the surface of loquat fruit.

[0048] Nanoparticle size and potential measurement First, the GP-NPs coating solution and the GP-MeJA@NPs coating solution were contacted with distilled water through an ion exchange membrane for one day to remove excess residual salt. Then, a certain amount of the nanoparticle coating solution was diluted 100 times with pure water. Finally, the nanoparticle solution was measured using a nanoparticle size and Zeta potential analyzer (Zetasizer Nano ZS90).

[0049] As shown in Table 1, the average particle sizes of NPs and MeJA@NPs were 135.13 nm and 328.9 nm, respectively, compared with SEM (…). Figure 2 The observed nanoparticles exhibited a consistent microstructure. With the addition of MeJA, the PDI of the nanoparticles increased from 0.135 to 0.21, indicating a relatively large diameter difference among the clustered MeJA@NPs. Figure 2 (b) Furthermore, the particle size uniformity of the MeJA@NPs solution is lower than that of the NPs solution. Simultaneously, the absolute value of the Zeta potential of the MeJA@NPs solution is greater than that of the NPs solution, indicating that its dispersion system is more stable. These results demonstrate that MeJA@NPs successfully encapsulates MeJA.

[0050] Table 1. Particle size, dispersion index, and potential of nanoparticles NPs 135.13±5.75 0.135±0.012 -17.70±0.91 MeJA@NPs 328.90±3.71 0.210±0.015 -23.83±0.58 a Mean particle diameter (MPD)r: Average particle size b PDI (polydispersity index): The index of polydispersity c Zeta Potential: Zeta potential.

[0051] Fourier transform infrared spectroscopy determination Using the Nicole iS50FTIR spectrometer (Thermo Scientific) TM Infrared analysis was performed on thin film samples and freeze-dried nanoparticles in the United States. The testing procedure was as follows: attenuated total reflectance method was used, and the instrument was set to a wavelength range of 4000 cm⁻¹. -1 ~400cm -1 The scanning resolution is 4cm. -1 The number of scans was 32.

[0052] like Figure 4 As shown in figure a, the characteristic peak of CMCS is 1586 cm⁻¹. -1 The absorption peak of the NH bending vibration at 1418 cm⁻¹-1 The absorption peak of the carboxyl stretching vibration at 1055 cm⁻¹ -1 The CO stretching vibration absorption peak at [location missing]. After crosslinking with CaCl2, the three absorption peaks of CMCS were enhanced in the nanoparticles. MeJA at 2961 cm⁻¹ -1 CH3- stretching vibration at 1733 cm -1 The absorption peaks of the pentacyclic ketones at that location all disappeared in the infrared spectrum of MeJA@NPs. For example... Figure 4 As shown in b, the NH bending vibration absorption peak (1586 cm⁻¹) of nanoparticles appeared in the GP-NPs and GP-MeJA@NPs coating films. -1 ) and the absorption peak of the carboxyl stretching vibration (1418 cm⁻¹) -1 ), and from the 6-glycosidic bond (922 cm) of pullulan. -1 Absorption peaks were observed in all three groups of coated film samples. These results indicate that MeJA@NPs successfully prepared and encapsulated MeJA, and could be fully mixed with GP to form a film for loading.

[0053] Thermal stability analysis was performed on the samples using a Mettler Toledo STARe System TGA2 instrument (Switzerland). The procedure was as follows: approximately 4 mg of sample was placed in the ceramic crucible of the instrument, and dry nitrogen gas was continuously introduced. The temperature range was set to 30℃–600℃, and the heating rate was set to 10℃ / min. -1 Record the weight loss curves for each sample.

[0054] like Figure 5 As shown, CMCS rapidly loses weight between 200℃ and 400℃, reaches its maximum pyrolysis rate at 280℃, and ultimately retains 45.6% at 600℃. Crosslinking CMCS with CaCl2 significantly improves the thermal stability of NPs, while MeJA@NPs exhibits a similar pyrolysis trend to NPs. The weight loss of the GP-coated film initially comes from the evaporation of bound water (50℃–150℃), followed by the rapid degradation of GL and PUL, ultimately leaving 16.1% at 600℃. The addition of nanoparticles improves the thermal stability of the coating film. The weight loss rate and weight loss of GP-NPs and GP-MeJA@NPs coated films are both lower than those of the GP-coated film between 200℃ and 300℃, ultimately retaining 30.9% at 600℃. These results indicate that the GP-MeJA@NPs coating possesses good thermal stability.

[0055] Nanoparticle encapsulation and sustained-release MeJA performance determination Encapsulation efficiency determination: The MeJA@NPs solution was centrifuged at 8000 rpm for 30 min, and 10 μL of the supernatant was taken. The free MeJA content in it was determined by GCMS (7000C, GC / MS Triple Quad, Agilent Technologies, USA) to determine the encapsulation efficiency of the nanoparticles. The calculation formula is as follows: where M i is the initial content of MeJA added in the MeJA@NPs solution, and M f is the free MeJA content in the solution. The MeJA content was calculated using the regression equation: y = 170998x - 9515.5, R 2 = 0.99, where y is the peak area and x is the MeJA content.

[0056] In vitro release determination of MeJA: The MeJA@NPs solution was aliquoted into ultrafiltration tubes, 20 mL per tube, for a total of 18 tubes (6 test time points, 3 replicates for each time point). It was centrifuged at 8000 rpm for 30 min to remove the supernatant, and the separated MeJA@NPs were stored at 0 °C. At each test point, three tubes of MeJA@NPs were randomly selected, 20 mL of ethanol solution was added to each tube, and the remaining MeJA in the nanoparticles was extracted by shaking for 24 h. After centrifugation, 10 μL of the supernatant was taken, and the MeJA content was analyzed by GCMS and the release curve was fitted using the Avrami equation.

[0057] Calculated by the encapsulation efficiency formula, the encapsulation efficiency of MeJA@NPs was 94.83%, which was about 7.5 times higher than that of the MeJA sustained-release composite film prepared by the inventor in the previous stage, further reducing the loss and waste of MeJA and improving its utilization efficiency. As Figure 6 shown, MeJA@NPs could continuously release MeJA for 30 days with a release rate of 81.79%, ensuring that loquat fruits could continuously receive the stimulation of MeJA during storage. The release curve was successfully fitted using the modified Avrami equation (n = 0.3746, k = 0.3776, R 2 = 0.875), which conformed to the diffusion-limited model (0.3 < n < 0.9), indicating that MeJA could be released from the solid-phase nanoparticles into the gaseous air.

[0058] Cytotoxicity test Cell culture and sample preparation Human gastric mucosal cells (GES-1, purchased from Saibakang Biotechnology) were cultured in a constant temperature incubator at 5% CO2 and 37 °C. The culture medium of GES-1 was prepared according to fetal bovine serum (164210-50, Procell): 1640 medium (10-010-CVRC, Corning) at a ratio of 1:9.

[0059] First, the GP-MeJA@NPs coated film sample to be tested was cut into 1×1cm pieces. 2 The cells were placed in cubes and irradiated under a UV lamp for 30 minutes before use. Next, GES-1 cells in the logarithmic growth phase were harvested, counted, and their concentration adjusted to 4 × 10⁻⁶ cells / mL. 4 Each well was inoculated into a 24-well plate containing culture medium (16 mm well). Finally, GP-MeJA@NPs coated film samples were added, and the plates were co-cultured in a 5% CO2, 37°C incubator for 24 h, with a blank control group (CK group) included. The experiment was set up with three replicates.

[0060] MTT assay for cell viability After co-culturing with the GP-MeJA@NPs coated film, the culture medium was removed, and each well was washed three times with phosphate buffer. 1 mL of solution containing 0.5 mg of phosphate buffer was added to each well. -1 MTT culture medium was incubated at 37°C with 5% CO2 for 4 hours. The supernatant was discarded, and 500 μL of dimethyl sulfoxide was added to each well. After gentle shaking for 10 min, 150 μL of the supernatant was transferred to a 96-well plate, and the absorbance at 570 nm was measured using a microplate reader. GES-1 cell relative viability % = 100% × (experimental group absorbance) / (control group absorbance).

[0061] Cell viability was determined using a live / dead cell staining kit (BB-4126, Shanghai Beibo Biotechnology). After co-culturing with GP-MeJA@NPs coated films, the culture medium was removed, the cells were washed with phosphate buffer, 1 mL of staining solution was added to each well, and the cells were incubated at room temperature in the dark for 15 min. The cells were then observed and photographed using a fluorescence microscope (Zeiss Axio).

[0062] Consumers may accidentally ingest coatings when consuming fruit; therefore, food safety and biocompatibility are key evaluation indicators for fruit coatings. MTT assays showed that the relative viability of GES-1 cells in the GP-MeJA@NPs coated film was 98.47% ± 1.64%, which was not significantly different from the CK group (100% ± 1.13%) (sig. > 0.05). Furthermore, electron microscopy using staining methods revealed that the GP-MeJA@NPs coated film treatment group exhibited high survival rate and good viability of GES-1 cells, with no significant difference from the CK group. Figure 7 The above results indicate that the GP-MeJA@NPs coating is non-toxic to human gastric mucosal cells. Furthermore, the raw material components of the GP-MeJA@NPs coating are all natural, biodegradable biopolymers with good biocompatibility. Therefore, GP-MeJA@NPs can be used as an in-situ coating for postharvest fruit preservation.

[0063] In summary, the MeJA slow-release nanoparticle coating exhibits excellent biocompatibility and food safety, making it suitable for post-harvest in-situ coating treatment of loquat fruit. The preparation of the MeJA slow-release nanoparticle coating addresses the issues of low encapsulation efficiency and trace acetic acid residue present in MeJA slow-release composite films. The encapsulation efficiency of the slow-release nanoparticles for MeJA is approximately 7.5 times higher than that of previous MeJA slow-release composite films, further improving MeJA utilization and reducing waste during preparation. Furthermore, due to the acidic solubility of chitosan, the inventors' previous MeJA slow-release composite films used a 2% acetic acid aqueous solution as a solvent, while the slow-release nanoparticle coating uses purified water, thus avoiding acetic acid residue and other issues. The coating also possesses hydrophilic properties, facilitating quick washing before consumption.

[0064] Experimental Preparation Example: Material Prepared for the Prevention and Control of Lignification Due to Chill Damage in Loquat Fruit Loquat fruits (Eriobotrya japonica Lindl.cv. Luoyangqing, 'Luoyangqing') were sourced from an orchard in Taizhou City, Zhejiang Province, and transported to the laboratory at Zhejiang University's Zijingang Campus on the same day of harvest. For sample processing, the fruits were first screened based on uniform size, color, and maturity, and the absence of mechanical damage or disease. Then, 15 fruits were randomly selected from the screened samples and immediately sampled on the day of harvest (0d). The remaining fruits were randomly divided into three batches of 45 fruits each for different treatment conditions. Specifically: the first batch of loquat fruits received no treatment and served as the control group (CK). The second batch of fruit samples underwent in-situ coating treatment, being immersed in a GP-NPs coating solution for 10 minutes and then air-dried. The third batch of fruit samples also underwent in-situ coating treatment, being immersed in a GP-MeJA@NPs coating solution for 10 minutes and then air-dried. Finally, all fruit samples from all groups were placed in non-sealed plastic boxes with ventilation openings on both sides and stored in a 0℃ cold storage.

[0065] On each sampling day (0d, 3d, 6d, and 12d from the harvest date), 15 fruits were randomly selected from each group and divided into 3 equal portions, serving as 3 biological replicates for that group's test day. The sampling process was as follows: the loquat peel and pit were removed, the pulp was cut into small pieces, frozen immediately with liquid nitrogen, and the pulp pieces were ground into powder using a grinder, then stored at -80℃ for later use.

[0066] The effect of slow-release nanoparticle coating treatment on inhibiting lignification caused by chilling injury in loquat fruit. like Figure 8As shown, during the entire storage period (0℃), the firmness of the loquat fruit in the CK group increased from 3.82N to 4.57N, and the lignin content increased from 0.146×10⁻⁶. 3 A280 kg -1 FW increased to 0.268×10 3 A280 kg -1 FW indicates the occurrence of lignification due to chilling injury in loquat fruit. Compared with the control group, the GP-MeJA@NPs coating treatment group showed a significant and sustained lignification inhibition effect (P<0.05), reducing lignin content accumulation by 56.9% throughout the storage period, and exhibiting the best inhibition of hardness increase on day 3, reducing it by 86.7%. The GP-NPs coating treatment without MeJA encapsulation did not alleviate chilling injury lignification in loquat fruit, and its hardness and lignin content were not significantly different from the control group. These results indicate that MeJA is a key active factor in inhibiting chilling injury lignification in loquat fruit.

[0067] Effects of slow-release nanoparticle coating treatment on phenylpropanoid metabolism in loquat fruit stored at low temperatures phenylpropane metabolic enzyme activity like Figure 9 As shown in Figure a, the PAL activity of loquat fruits in each group showed a trend of first increasing and then decreasing during the entire storage period, and there was no significant difference between the groups, indicating that neither GP-NPs nor GP-MeJA@NPs coating treatment affected the PAL activity of 'Luoyangqing' loquat fruits.

[0068] like Figure 9 As shown in bc, the C4H and 4CL activities of loquat fruits in all groups showed an increasing trend throughout the storage process. Compared with the CK group, the GP-MeJA@NPs coating treatment group significantly enhanced the activities of C4H and 4CL (P<0.05), while the GP-NPs coating treatment group showed no significant difference.

[0069] like Figure 9 As shown in Figure d, the POD activity in the CK group increased rapidly on day 3 and then remained at a high level throughout the storage period. The POD activity of the GP-NPs coated group showed the same trend as the CK group, and there was no significant difference between the groups. GP-MeJA@NPs coating treatment significantly slowed the increase of POD on day 3 and significantly inhibited POD activity in the later stages of storage by continuously releasing MeJA (P<0.05). Figure 8 b. Correlation analysis revealed a positive correlation between POD activity and lignin content in loquat fruit (PCCs). POD =0.641, sig.<0.05).

[0070] In summary, the MeJA slow-release nanoparticle coating may achieve the effect of slowing down the lignification of loquat fruit after cold damage by releasing MeJA to inhibit POD activity, while enhancing the activity of C4H and 4CL in the fruit during storage.

[0071] phenylpropane metabolites like Figure 10 As shown, GP-MeJA@NPs coating treatment promoted the accumulation of total phenols and total flavonoids in loquat fruit and maintained a high level throughout storage (P<0.05, compared with CK). The GP-NPs coating group showed no significant difference from the CK group and exhibited an increasing trend. These results indicate that the GP-MeJA@NPs coating with slow-release capability can induce the accumulation of total phenols and total flavonoids in fruit by releasing MeJA, thereby improving the fruit's antioxidant capacity and better coping with low-temperature stress.

[0072] Effects of slow-release nanoparticle coating treatment on oxidative metabolism of loquat fruit stored at low temperatures Oxidative metabolic enzyme activity like Figure 11 As shown in Figure a, the SOD activity of loquat fruits in the GP-NPs coating treatment group showed an overall decreasing trend throughout the storage period, consistent with the CK group. The GP-MeJA@NPs coating treatment effectively inhibited the decrease in SOD activity and maintained a higher SOD activity than the CK group throughout the storage period (P<0.05). SOD is the first line of defense in oxidative metabolism; a decrease in its activity leads to the fruit's inability to effectively scavenge O2. - ROS levels are detrimental to maintaining the oxidative metabolic balance within the fruit.

[0073] CAT activity in CK group and GP-NPs coated group ( Figure 11 b) The activity showed a slow upward trend with no significant difference between the two groups; however, after GP-MeJA@NPs coating treatment, the CAT activity of the fruit increased rapidly and remained at a high level throughout the storage period (P<0.05, compared with the CK group), which was beneficial for the fruit to quickly remove H2O2.

[0074] APX activity in loquat fruits of each treatment group ( Figure 11 c) The overall trend was upward. The GP-MeJA@NPs coating treatment significantly enhanced the APX activity of the fruit in the early stage of low temperature storage (P<0.05, compared with the CK group).

[0075] Oxidative metabolites like Figure 12As shown in Figure a, there was no significant difference in H2O2 content among the groups in the early stage of storage. However, with the increase of storage time, the H2O2 content of the CK group and the GP-NPs coating treatment group showed the same upward trend, while the GP-MeJA@NPs coating treatment group showed a downward trend. The results indicate that the MeJA slow-release nanoparticle coating effectively reduced the H2O2 content (P<0.05), which may be because the MeJA released by the slow-release coating enhanced the activity of SOD, CAT, and APX in the fruit, and effectively removed H2O2 through the synergistic effect of reactive oxygen species scavenging enzymes.

[0076] like Figure 12 As shown in b, the MDA content in the CK group increased from 32.63 nmol g. -1 FW increased to 43.63 nmol g -1 FW indicates that the loquat fruit is suffering from chilling injury and that the cell membranes are damaged. There was no significant difference in MDA between the GP-NPs coated group and the CK group. Compared to the CK group, the GP-MeJA@NPs coating treatment significantly reduced MDA production by 88.7% during storage (P<0.05), effectively alleviating chilling injury symptoms in the fruit.

[0077] proline content like Figure 13 As shown, the Pro content in the CK group and the GP-NPs coating treatment group did not show significant trends throughout the storage process, and there were no significant differences within or between the two groups. The GP-MeJA@NPs coating treatment induced rapid Pro accumulation in loquat fruits, with its content consistently higher than that in the CK group throughout the storage period (P<0.05), reaching its highest level on day 6. Pro accumulation can improve the fruit's cold resistance, thereby mitigating chilling injury.

[0078] The above results of oxidative metabolic enzymes indicate that the GP-MeJA@NPs coating can enhance the activity of SOD, CAT and APX during fruit storage by continuously releasing MeJA, thereby maintaining the oxidative metabolic balance in loquat fruit and reducing oxidative damage to the fruit.

[0079] In summary, GP-NPs coating treatment without MeJA loading cannot alleviate lignification caused by chilling injury in loquat fruit, nor does it affect the fruit's phenylpropane metabolism and oxidative metabolism.

[0080] Furthermore, cost calculations were performed on two slow-release packaging materials. The results showed that the cost of treating a single loquat fruit with the inventor's previous slow-release composite film (a composite film formed by mixing gelatin, pullulan, chitosan, and MeJA) was approximately RMB 0.73, while the cost of the slow-release nanoparticle coating was approximately RMB 0.05, only about 6.8% of the former. Meanwhile, considering the higher MeJA encapsulation efficiency of the coating treatment (94.83% vs. 11.05%), it can be considered that the fruit coating treatment has a greater advantage than the film treatment in terms of reducing the cost and increasing efficiency of MeJA usage. On the other hand, the current market price of a single loquat fruit is approximately RMB 1.3 (approximately 36g per fruit, based on 2023 purchase price), and the processing cost of this invention is only 3.8% of its selling price. Therefore, the coating treatment technology developed in this invention has potential market application value.

[0081] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. A method for inhibiting lignification of loquat fruit due to postharvest chilling injury, characterized in that, Includes the following steps: (1) Preparation of MeJA@NPs nanoparticle solution: MeJA was added to a carboxymethyl chitosan solution to form a mixed solution of carboxymethyl chitosan and MeJA. Under stirring conditions, CaCl2 solution was added dropwise to the mixed solution of carboxymethyl chitosan and MeJA at a uniform rate and mixed well to form a carboxymethyl chitosan nanoparticle solution loaded with MeJA, namely MeJA@NPs nanoparticle solution. (2) Preparation of GP-MeJA@NPs coating solution: Gelatin and pullulan were dissolved in water to form a mixed solution. Then, glycerol was added and stirred until homogeneous to form a GP coating solution. The GP coating solution was mixed with an equal volume of MeJA@NPs nanoparticle solution to obtain a GP-MeJA@NPs coating solution. (3) In-situ coating processing: Immerse loquat fruits in GP-MeJA@NPs coating solution at room temperature for 10-15 minutes, then remove and air dry; In step (1), the volume ratio of CaCl2 solution to the mixed solution of carboxymethyl chitosan and MeJA is 1:2-3; In step (1), the mixed solution of carboxymethyl chitosan and MeJA has the following concentrations: carboxymethyl chitosan concentration of 0.01-0.02 g / mL and MeJA concentration of 25-32 μmol / L.

2. The method according to claim 1, characterized in that, The volume ratio of CaCl2 solution to a mixed solution of carboxymethyl chitosan and MeJA is 1:2.

5.

3. The method according to claim 1, characterized in that, In a mixed solution of carboxymethyl chitosan and MeJA, the concentration of carboxymethyl chitosan was 0.01 g / mL and the concentration of MeJA was 28 μmol / L.

4. The method according to claim 1, characterized in that, In step (1), the concentration of CaCl2 solution is 0.01-0.02 g / mL.

5. The method according to claim 1, characterized in that, In step (1), the CaCl2 solution is added at a rate of 3-5 mL / min.

6. The method according to claim 1, characterized in that, In step (2), the volume concentration of glycerol in the GP coating solution is 0.5-1%, and the total concentration of gelatin and pullulan is 0.01-0.02 g / mL.

7. The method according to claim 6, characterized in that, The mass ratio of gelatin to pullulan is 1-2:1.

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