Application of linalool in prevention and treatment of pathogenic bacteria and fruit fresh-keeping coating agent

Abstract

This invention discloses the application of linalool in the control of pathogens and its fruit preservation coating agent. This invention is the first to discover that linalool has a significant control effect on pathogens of citrus fruits, especially on *Penicillium italicum*, the main pathogen causing post-harvest rot of citrus fruits, providing a new and safe agent for the preservation of citrus fruits. Furthermore, linalool is prepared into a coating agent for post-harvest treatment of citrus fruits, which can effectively prevent rot caused by pathogens, extend the storage period, and ensure excellent fruit appearance and marketability.

Description

Technical Field

[0001] This invention relates to the field of fruit and vegetable preservation technology, specifically to the application of linalool in the prevention and control of pathogens and its fruit preservation coating agent. Background Technology

[0002] Citrus (Citrus), belonging to the Rutaceae family and the Citrus genus, is one of the world's major fruit crops, primarily grown in countries such as China, Brazil, and the United States. Currently, my country ranks among the world's top countries in terms of citrus planting area and yield. Citrus fruits are delicious and rich in active ingredients such as vitamin C, polyphenols, and flavonoids, making them very popular with consumers. Citrus fruits have a concentrated ripening period, high yields, and are mainly sold fresh. During harvesting, storage, transportation, and sales, they face numerous threats from pathogens. Once infected, the fruit will suffer varying degrees of damage and spoilage, resulting in significant economic losses. With advancements in variety development, more and more citrus varieties have emerged, leading to a substantial increase in yield. Therefore, it is necessary to develop post-harvest disease control technologies for citrus to minimize disease incidence, extend shelf life, and deliver high-quality fruit to consumers.

[0003] Timely post-harvest treatment can effectively suppress post-harvest diseases in citrus fruits and maintain their quality. Currently, the industry's post-harvest treatment technologies for citrus fruits mainly include physical preservation (such as cold storage, ozone storage, and controlled atmosphere storage) and chemical preservation (chemically synthesized preservatives such as preservatives and fungicides). While chemical fungicides are readily available and easy to use, long-term use may lead to drug resistance in pathogens and pose potential pollution risks to human health and the environment. Considering cost and promotional effects, low-temperature storage remains the primary choice for post-harvest treatment in the industry; however, a complete cold storage and cold chain quality control logistics preservation system has not yet been established. Therefore, researching and promoting new, green, and natural preservatives under ambient temperature storage conditions has significant economic value and environmental protection significance.

[0004] Citrus fruits are susceptible to various pathogenic fungi after harvest, the most common being Penicillium italicum (… Penicillium italicum Penicillium, anthrax ( ) caused by Penicillium, anthrax ( Colletotrichum gloeosporioides Anthrax caused by Penicillium fingerlingum ( ) Penicillium digitatum Green mold caused by citrus sour rot fungus and citrus acid rot fungus ( Geotrichum citri- aurantii This includes diseases such as sour rot caused by Penicillium mold. Penicillium mold is one of the main diseases causing post-harvest rot in citrus fruits. The conidia of its pathogen, *Penicillium italicum*, are resistant to low temperatures, making it more susceptible to disease and capable of infecting intact fruits. Therefore, the control of *Penicillium italicum*, the pathogen causing Penicillium mold, is extremely important.

[0005] Linalool, 2,6-dimethyl-2,7-octadien-6-ol, is a naturally occurring acyclic monoterpene found in a variety of aromatic plants and widely distributed in over 200 essential oils worldwide. Due to the chiral center at the third carbon position in its molecular structure, it exists as two enantiomers, each with a different aroma depending on its source. Linalool possesses analgesic, anti-anxiety, sedative-hypnotic, and relatively high safety profiles. In my country's national food safety standard GB 1886-2015, linalool is listed as a food additive.

[0006] Currently, linalool is widely used in the chemical industry, fragrances, and flavorings. In its application to fruit and vegetable preservation, it is mostly found as a major component of essential oils. There are few reports of linalool being used directly for the prevention and control of citrus fruit diseases. Summary of the Invention

[0007] The purpose of this invention is to address the above-mentioned problems by providing the application of linalool in the control of protozoa and its fruit preservation coating agent.

[0008] To achieve its objective, the present invention employs the following technical solution: The first aspect of the present invention provides the use of linalool in any of the following: (1) Use in inhibiting plant pathogens or in preventing and controlling plant diseases caused by said plant pathogens; (2) Use in the preparation of products that inhibit plant pathogens or prevent plant diseases caused by said plant pathogens; The plant pathogens include Penicillium italicum, Penicillium fingernail, or anthracnose.

[0009] Preferably, the plant is a citrus fruit. Preferably, in the above application technology solution, the working concentration of linalool is ≥3.2 μL / mL, more preferably ≥6.4 μL / mL, and even more preferably 3.2~26 μL / mL.

[0010] A second aspect of the present invention provides a fruit preservation coating agent comprising the following components: The mixture comprises 0.40-0.80% (v / v) chitosan co-solvent, 1.0-2.0% (w / v) chitosan, 1.0-2.0% (w / v) pullulan, 0.10-0.20% (v / v) linalool, and linalool co-solvent, wherein the mass of the linalool co-solvent is 0.6-1.5% of the mass of linalool, with the balance being water; the chitosan co-solvent is selected from glacial acetic acid, hydrochloric acid, or formic acid, and the linalool co-solvent is selected from methanol, ethanol, or Tween 80; preferably, it contains the following components: 0.40~0.60% (v / v) or 0.45~0.55% (v / v) chitosan co-solvent, 1.3~1.8% (w / v) chitosan, 1.3~1.8% (w / v) pullulan, 0.13~0.18% (v / v) linalool, linalool co-solvent, wherein the mass of the linalool co-solvent is 0.8~1.2% of the mass of linalool; or, 0.5% (v / v) chitosan cosolvent, 1.5% (w / v) chitosan, 1.5% (w / v) pullulan, 0.15% (v / v) linalool, linalool cosolvent, wherein the mass of the linalool cosolvent is 1.0% of the mass of linalool.

[0011] The third aspect of the present invention provides a method for preparing the above-mentioned fruit preservation coating agent, comprising the following steps: weighing each component in proportion, mixing, stirring and mixing thoroughly to obtain the product.

[0012] Preferably, the preparation method of the above-mentioned fruit preservation coating agent includes the following steps: adding chitosan co-solvent and chitosan to water and stirring thoroughly to dissolve and mix evenly to obtain a chitosan solution; adding pullulan polysaccharide to water and stirring thoroughly to dissolve and mix evenly to obtain a pullulan polysaccharide solution; adding linalool co-solvent to linalool and stirring thoroughly to mix evenly; mixing the prepared chitosan solution, pullulan polysaccharide solution, and linalool containing linalool co-solvent and stirring thoroughly to obtain the final product.

[0013] A fourth aspect of the present invention provides an antibacterial film for fruit preservation, the antibacterial film comprising the following components in a mass ratio of 14-18:14-18:1, wherein chitosan, pullulan, and linalool are present, preferably in a mass ratio of 15-17:15-17:1 or 16.2-17.2:16.2-17.2:1.

[0014] A fifth aspect of the present invention provides a film-forming solution for an antibacterial film used for fruit preservation, the film-forming solution comprising the following components in the following proportions: 0.40~0.80% (v / v) chitosan co-solvent, 0.5~1.0% (w / v) chitosan, 0.5~1.0% (w / v) pullulan, 0.03~0.06% (w / v) linalool, and 0.40~0.80% (v / v) linalool co-solvent; wherein the chitosan co-solvent is selected from glacial acetic acid, hydrochloric acid, or formic acid, and the linalool co-solvent is selected from methanol, ethanol, or Tween 80; Preferably, the film-forming solution comprises the following components in the following proportions: 0.40~0.60% (v / v) or 0.45~0.55% (v / v) chitosan co-solvent, 0.7~0.8% (w / v) chitosan, 0.7~0.8% (w / v) pullulan, 0.04~0.05% (w / v) linalool, and 0.40~0.60% (v / v) or 0.45~0.55% (v / v) linalool co-solvent; or: 0.50% (v / v) chitosan co-solvent, 0.75% (w / v) chitosan, 0.75% (w / v) pullulan, 0.045% (w / v) linalool, and 0.5% (v / v) linalool co-solvent.

[0015] The sixth aspect of the present invention provides a method for preparing the above-mentioned antibacterial film for fruit preservation, wherein each raw material component is weighed in proportion, mixed and stirred evenly, placed in a film-forming mold, and heated to evaporate the solvent to obtain an antibacterial film. The preferred preparation method includes the following steps: adding chitosan co-solvent and chitosan to water and stirring thoroughly to dissolve and mix, thereby obtaining a chitosan solution; adding pullulan polysaccharide to water and stirring thoroughly to dissolve and mix, thereby obtaining a pullulan polysaccharide solution; adding linalool co-solvent to linalool and mixing thoroughly; mixing the prepared chitosan solution, pullulan polysaccharide solution, and linalool containing linalool co-solvent, and mixing thoroughly to obtain a film-forming solution; placing the film-forming solution in a flat film-forming mold and allowing it to stand at 35~50℃ (preferably 40~45℃) for 20~28h to evaporate the solvent, thereby obtaining an antibacterial film.

[0016] The seventh aspect of the present invention provides the application of the above-mentioned fruit preservation coating agent, antibacterial film, or film-forming solution in fruit preservation; The preferred method for applying the fruit preservation coating agent is as follows: fully wet the sponge with the fruit preservation coating agent, and then use the sponge to apply the coating agent to the outer surface of the fruit until the outer surface of the fruit is evenly wetted by the coating agent. The preferred method of applying the antibacterial film is to wrap the fruit in the antibacterial film before storing / transporting it; Preferably, the fruit is a citrus fruit. The coating agent inhibits / kills pathogens carried by the fruit and prevents the fruit from rotting due to pathogens. The pathogens include Penicillium italicum, Penicillium fingernail, or anthracnose.

[0017] The beneficial effects of this invention are: It is the first discovery that linalool has a significant control effect on pathogens of citrus fruits, especially on *Penicillium italicum*, the main pathogen causing postharvest rot of citrus fruits, providing a new and safe agent for the preservation of citrus fruits. Furthermore, linalool can be prepared as a coating agent for postharvest coating treatment of citrus fruits, effectively preventing rot caused by pathogens, extending shelf life, and ensuring excellent fruit appearance and marketability. Attached Figure Description

[0018] Figure 1 This is the result of the inhibitory effect of linalool on common pathogens.

[0019] Figure 2 Results of the PDA plate inhibition experiment of linalool against Penicillium italicum.

[0020] Figure 3 These are the results of a storage experiment on summer oranges after coating.

[0021] Figure 4 This is the result of Fourier transform infrared spectroscopy analysis of the thin film using omics.

[0022] Figure 5 This is the result of XRD analysis of the thin film structure.

[0023] Figure 6 These are the results of the thin film mechanical property analysis.

[0024] Figure 7 These are the results of thin film microstructure analysis. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Unless otherwise specified, the methods described in the following embodiments are conventional methods.

[0026] Example 1: Antibacterial experiment of linalool against Penicillium italicum 1. Materials and Reagents 1.1 Main Experimental Reagents Linalool (CAS No.: 78-70-6): purity 98%; PDA medium (potato dextrose agar medium), PDB medium (potato dextrose broth medium).

[0027] 1.2 Materials and Processing Common postharvest pathogens of citrus: *Penicillium italicum* was collected separately (… Penicillium italicum ), Penicillium finger ( Penicillium digitatum Anthrax bacteria ( Colletotrichum gloeosporioides ), Citrus acid rot fungus ( Geotrichum citri-aurantii The bacterial strain was activated and then cultured in a constant temperature incubator at 28℃ for 5 days.

[0028] 96-well plate experiment: PDB solutions containing different concentrations of linalool were prepared. The different linalool concentrations were set as follows: 0, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, and 25.6 μL / mL. 100 μL of linalool-PDB solution and 100 μL of pathogenic spore suspension (1×10⁻⁶) were added to each well. 6 (number / mL). Incubate at 28℃ for 2 days.

[0029] PDA plate experiment: PDA plates containing different concentrations (0, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 μL / mL) of linalool were prepared. The spore suspension of *Penicillium italicum* was obtained by washing with sterile water (1×10⁻⁶ μL / mL). 6 (Spots / mL), place an 8mm×8mm sterile blank paper disc in the center of the plate, add 20 μL of Italian Penicillium spore suspension on the paper disc, and incubate at 28℃ for 7 days.

[0030] 2. Measurement Indicators and Methods 2.1 Inhibitory effect of linalool on common pathogens The inhibitory effect of linalool on common postharvest pathogens of citrus was determined using the broth dilution method. Different concentrations of linalool-PDB solution were added to 96-well plates. For each well in the drug treatment group, 100 μL of linalool-PDB solution was added. Two rows of wells were set up as controls: one row of wells containing blank PDB without linalool as a positive control, and one row of wells containing sterile water as a negative control. 100 μL of diluted spore suspensions (1×10⁻⁶) of the four common postharvest pathogens of citrus were also used. 6 Add (number / mL) to the corresponding well.

[0031] 2.2 Inhibitory effect of linalool on Penicillium italicum The inhibitory effect of linalool on Penicillium italicum mycelium was determined using the growth rate method. PDA plates were observed for 7 days, and photographs were taken starting from the second day. The colony diameter was measured every 24 hours using the cross-sectional method. The relative inhibition rate of mycelial growth, the virulence regression equation, and the half-maximal effective concentration (EC50) were calculated from the diameter.

[0032] Relative inhibition rate / % = (Coronavirus diameter in blank group - Coronavirus diameter in treatment group) / Coronavirus diameter in blank group × 100 Virulence regression equation: y=a+bx (in the virulence regression equation, x is the logarithm of the concentration, y is the probability value of the inhibition rate, and the EC50 value is the concentration required to achieve a 50% inhibition effect) 2.3 Determination of the minimum inhibitory concentration and minimum bactericidal concentration of Penicillium italicum PDA plates were cultured for 7 days, and the growth of *Penicillium italicum* on the plates was observed daily. The minimum inhibitory concentration (MIC) was the lowest concentration of linalool that could inhibit the growth of *Penicillium italicum* after 2 days, and the minimum fungicidal concentration (MFC) was the lowest concentration that would prevent the growth of *Penicillium italicum* mycelia after 7 days.

[0033] 3 Results 3.1 Inhibitory effect of linalool on common pathogens Cultures of four common postharvest pathogens of citrus in 96-well plates revealed that linalool only inhibited *Penicillium italicum*. The 96-well plates cultured for two days showed... Figure 1 As shown, at the same concentration of 3.2 μL / mL, only *Penicillium italicum* was inhibited from growing. Citrus acid rot fungi all grew normally at high concentrations of linalool (25.6 μL / mL); *Anthracis* and *Penicillium digitatum* still grew normally at a concentration of 3.2 μL / mL, and linalool only showed some inhibitory effect on *Anthracis* and *Penicillium digitatum* at a concentration of 6.4 μL / mL.

[0034] 3.2 Inhibitory activity and antibacterial effect of linalool against Penicillium italicum like Figure 2 As shown, PDA plate inhibition experiments were conducted on Penicillium italicum using different concentrations of linalool. On day 5, the average colony diameter in the MIC group was 13.75 mm, while the average colony diameter in the control group was 36.58 mm. The relative inhibition rate of the 3.2 μL / mL linalool treatment group was 62.41%.

[0035] The toxicity regression equation is y = 0.4090 + 3.0784x, and the correlation coefficient is 0.975.

[0036] The EC50 of linalool against *Penicillium italicum* was 1.358 μL / mL. The MIC was 3.2 μL / mL, and the MFC was 6.4 μL / mL.

[0037] 4. Conclusion Antimicrobial experiments using linalool on four major postharvest pathogens of citrus revealed that linalool only showed a strong inhibitory effect against *Penicillium italicum*. On PDA plates, after 5 days of incubation, linalool at a concentration of 3.2 μL / mL showed an inhibition rate of 62.41% against *Penicillium italicum*. The MIC (micronizable value) of linalool against *Penicillium italicum* was 3.2 μL / mL, and the MFC (micro-value-added value) was 6.4 μL / mL.

[0038] Example 2: Experiment on Penicillium infestation of Citrus aurantium fruit 1. Materials and Reagents 1.1 Materials and Processing Experimental materials: Summer oranges and golden oranges Experimental reagents: chitosan (CAS No. 9012-76-4), pullulan (CAS No. 9057-02-7), linalool (purity 98%).

[0039] I. Experiment on the infection of orange peel Prepare the coating agent: Prepare the coating agents listed in Table 1.

[0040] The Jincheng oranges were harvested in December 2024. After harvesting, they were transported back to the Citrus Research Institute of Southwest University. Fruits of uniform size, consistent ripeness, and free from pests, diseases, and mechanical damage were selected, washed, and disinfected with 75% alcohol. Then, an Italian Penicillium inoculation experiment was conducted: a 2mm diameter, 2mm length needle was used to create an artificial wound (2mm diameter, 2mm depth) in the orange fruit, and 5 μL of Italian Penicillium spore suspension (1×10⁻⁶) was injected into the wound. 6 (per fruit / mL). After air-drying for 2 hours, the fruits were randomly grouped into groups of 20 fruits each. The prepared coating agent was then applied: the sponge was soaked in the prepared coating agent solution until it was fully absorbed, and then the sponge was used to apply the coating agent to the outer surface of the fruit (the outer surface of the fruit should be evenly moistened with the coating agent).

[0041] After the coating was dried in the shade, the fruit was stored in a culture room at 28℃ and 85% relative humidity. Photos were taken and observed from day 2 to day 7. The diameter of lesions on the fruit peel was measured after disease onset. The obtained experimental data were statistically analyzed and significance was determined using Microsoft Excel 2003 (P<0.05).

[0042] Table 1. Experimental treatment scheme for Penicillium citrinum infection.

[0043] The preparation methods of the coating agents for each treatment group are as follows: (1) Groups K1 and K2: To prepare a 1% (v / v) glacial acetic acid solution as a solvent: Slowly add 10 mL of glacial acetic acid (99% purity) dropwise to 990 mL of pure water and mix thoroughly. Chitosan is almost insoluble in water but readily soluble in dilute acid solutions with a pH below 6, such as hydrochloric acid, formic acid, and acetic acid.

[0044] K1: Using 1% (v / v) glacial acetic acid as a solvent, dissolve chitosan in the 1% (v / v) glacial acetic acid solution and stir until dissolved to prepare a coating agent solution with a chitosan concentration of 1.5% (w / v). K2: Using 1% (v / v) glacial acetic acid as a solvent, dissolve chitosan in the 1% (v / v) glacial acetic acid solution and stir until dissolved to prepare a coating agent solution with a chitosan concentration of 3% (w / v).

[0045] (2) Groups P1 and P2: P1: Dissolve pullulan in pure water to prepare a coating agent solution with a pullulan concentration of 1.5% (w / v); P2: Dissolve pullulan in pure water to prepare a coating solution with a pullulan concentration of 3% (w / v).

[0046] (3) Groups PF1 and PF2: PF1: Dissolve 15g pullulan in 1L of pure water using magnetic stirring until completely dissolved to obtain a 1.5% (w / v) pullulan solution. Add 0.02g Tween 80 to 2g linalool and vortex to mix. Then, slowly add 1.5mL of linalool containing Tween 80 to the prepared 1L pullulan solution and mix well to obtain a coating solution containing 1.5% (w / v) pullulan and 0.15% (v / v) linalool. PF2: Dissolve 30g pullulan in 1L of pure water using magnetic stirring until completely dissolved, obtaining a 1.5% (w / v) pullulan solution. Add 0.02g Tween 80 to 2g linalool and vortex to mix. Then, slowly add 1.5mL of linalool containing Tween 80 to the prepared 1L pullulan solution and mix well to obtain a coating agent solution containing 3% (w / v) pullulan and 0.15% (v / v) linalool.

[0047] Tween 80, as a co-solvent for linalool, solves the hydrophobicity problem of linalool, making the solution system more uniform and stable. Methanol or ethanol can also be used instead of Tween 80.

[0048] (4) Groups KF1 and KF2: KF1: Weigh 15g of chitosan and slowly add it to 1L of 1% (v / v) glacial acetic acid. Stir until the solution is completely transparent to obtain 1L of 1.5% (w / v) chitosan solution for later use. Add 0.02g of Tween 80 to 2g of linalool and vortex to mix. Then, slowly add 1.5mL of linalool containing Tween 80 to the prepared 1L chitosan solution and mix thoroughly to obtain a coating agent solution containing 1.5% (w / v) chitosan and 0.15% (v / v) linalool.

[0049] KF2: Following the preparation method of KF1, a coating agent solution containing 3% (w / v) chitosan + 0.15% (v / v) linalool was prepared.

[0050] (5) KPF1 and KPF2 groups: KPF1: Take 10 mL of glacial acetic acid and stir magnetically in 990 mL of pure water to obtain a glacial acetic acid solution. Weigh 30 g of chitosan and slowly add it to the glacial acetic acid solution until the solution is completely transparent to obtain solution 1. Take 30 g of pullulan and stir in 1 L of pure water until completely dissolved to obtain solution 2. Add 0.02 g of Tween 80 to 2 g of linalool and vortex mix. Measure 500 mL of solution 1 and 500 mL of solution 2 and mix them evenly to obtain a mixture. Add 1.5 mL of linalool containing Tween 80 to the mixture and stir evenly to obtain a coating agent solution containing 1.5% (w / v) chitosan + 1.5% (w / v) pullulan + 0.15% (v / v) linalool.

[0051] KPF2: Take 10 mL of glacial acetic acid and stir magnetically in 990 mL of pure water to obtain a glacial acetic acid solution. Weigh 60 g of chitosan and slowly add it to the glacial acetic acid solution until the solution is completely transparent to obtain solution 1. Take 60 g of pullulan and stir in 1 L of pure water until completely dissolved to obtain solution 2. Add 0.02 g of Tween 80 to 2 g of linalool and vortex mix. Measure 500 mL of solution 1 and 500 mL of solution 2 and mix them evenly to obtain a mixture. Add 1.5 mL of linalool containing Tween 80 to the mixture and stir evenly to obtain a coating agent solution containing 3% (w / v) chitosan + 3% (w / v) pullulan + 0.15% (v / v) linalool.

[0052] II. Summer Orange Storage Experiment Meanwhile, based on the optimal treatment group KPF1 obtained from previous peel infection experiments, a storage experiment was conducted on summer oranges. Summer oranges were harvested from the orchard in June 2025 and transported to the storage warehouse on the same day. Fruits of uniform size, consistent maturity, and free from pests, diseases, and mechanical damage were selected and sorted into different boxes for treatment. The coating solution in Table 2 was used for treatment: the fruit was immersed in the coating solution for 10 minutes, then removed, air-dried naturally, and then transferred to a cold storage at 4-6℃ and 80-85% RH for 90 days. Fruit quality was tested every 30 days.

[0053] Table 2 Summer Orange Storage Experimental Treatment Scheme

[0054] 2. Measurement Indicators and Methods 2.1 Determination of pericarp infection inhibition rate Measurements were taken starting with the diameter of obvious lesions on the fruit peel. The disease incidence was observed daily in each group, and the diameter of the lesions on each fruit was measured. The formulas for calculating the fruit peel disease inhibition rate and relative inhibition rate are as follows: Fruit peel disease inhibition rate (%) = ((diameter of single fruit lesion in blank group - diameter of single fruit lesion in treatment group) / diameter of single fruit lesion in blank group) × 100; Relative inhibition rate of fruit peel (%) = ((diameter of single fruit lesion in treatment group 1 - diameter of single fruit lesion in treatment group 2) / diameter of single fruit lesion in treatment group 1) × 100.

[0055] 2.2 Determination of Fruit Weight Loss Rate Citrus fruits lose water through transpiration via stomata after harvest, and also consume dry matter through respiration. Weight loss rate represents the total loss of water and dry matter from citrus fruits and can be used to assess the effectiveness of coatings. Fifteen fruits were randomly selected and weighed upon arrival at storage, and weighed again every 30 days. The formula for calculating the weight loss rate is as follows: Weight loss rate (%) = ((Weight of a single fruit upon entry into storage - Weight of a single fruit at the corresponding time point) / Weight of a single fruit upon entry into storage) × 100 2.3 Determination of Fruit Rot Rate Citrus fruits can become diseased during storage due to pathogen infection, resulting in varying degrees of damage to the peel, an unpleasant odor, and loss of commercial value. In the statistical analysis, any citrus fruit showing obvious signs of disease was classified as rotten. Ninety citrus fruits were randomly selected from each treatment and divided into three groups. The extent of fruit rot was assessed every 30 days.

[0056] "Obvious lesions" refer to any typical visible symptoms caused by pathogenic microorganisms that are observed directly on the surface of citrus fruits (including the stem and navel) by the naked eye. The main characteristics include, but are not limited to: (1) the appearance of specific lesions: such as brown, black or water-soaked rotten patches. (2) the production of mold or mold spots: such as white mycelium, or spore layers of blue, green or other colors. (3) changes in texture: the tissue at the lesion site is obviously softened, sunken or water-soaked.

[0057] The formula for calculating rotten fruit is: Rot rate (%) = (Number of rotten fruits / Total number of treated fruits) × 100.

[0058] 2.4 Sensory evaluation of fruit storage A panel of five professionals was assembled. In the sensory evaluation room, the judges assessed the freshness of the peel, distinctive aroma, moisture content, sweet-acid balance, off-flavors, and texture, assigning a total score from 1 to 9, with 5 indicating average (commercially acceptable) quality. Each judge's score was collected for data analysis.

[0059] 3 Results 3.1 Effects of different coating agents on postharvest infection of orange peel The effects of different coating agents on the growth of fungal spots on the peel of oranges infected with Penicillium italicum were observed. The inhibition rate was calculated by measuring the diameter of the spots on day 7.

[0060] The results are shown in Table 3. All different coating agents effectively inhibited the growth of *Penicillium italicum*, with KPF1 showing the best effect in inhibiting lesion growth. Among the different coating agents, the coating agent containing 3% chitosan produced a thicker film after application. Compared to fruits treated with other coating agents, fruits treated with pullulan + linalool alone had a more noticeable "gray" layer on the surface, resulting in poorer marketability.

[0061] Table 3. Effects of different treatments on the inhibition of postharvest lesions in oranges.

[0062] In summary, the KPF1 group containing 1.5% chitosan, 1.5% pullulan, and 0.15% linalool effectively inhibited Penicillium. Compared with the blank group (47.45±3.65 mm in diameter), the lesion diameter in the KPF1 treatment group was only 25.6±6.06 mm, with an inhibition rate of 46.05%.

[0063] Experiments have shown that, for example Figure 3 As shown, the KPF1 treatment group effectively inhibited the growth of Penicillium while giving the fruit peel a glossy appearance. The pure chitosan treatment groups (K1 and K2) formed a distinct membrane on the peel surface, while the pure pullulan treatment groups (P1 and P2) had a poorer peel color compared to other groups. The KPF1 group showed the best inhibitory effect on postharvest Penicillium infection in oranges, while maintaining better fruit appearance quality.

[0064] 3.2 Results of Summer Orange Storage Experiment 3.2.1 Weight loss rate of summer oranges Table 4 shows the changes in weight loss rate of summer oranges treated with different coating agents during storage. The weight loss rate of the KPF1 group was significantly lower than that of the CK group throughout the process.

[0065] Table 4. Results of weight loss during storage of summer orange fruits after postharvest coating treatment.

[0066] 3.2.2 Summer orange fruit decay rate The changes in fruit rot rate are shown in Table 5 below. After 90 days of storage, the rot rate of the CK group was 9%, and the rot rate of the KPF1 group was 3.33%. The rot rate of the KPF1 group was 63.33% lower than that of the CK group, and the rot rate of the fruit in the KPF1 group was significantly lower than that in the CK group.

[0067] Table 5 Results of postharvest storage decay rate of summer orange fruit

[0068] 3.2.3 Sensory evaluation of summer orange fruit Sensory evaluations of citrus fruits were conducted at 30, 60, and 90 days. Throughout the process, the sensory evaluation scores of the KPF1 group were higher than those of the CK group, and the score remained greater than 5 at 90 days, indicating commercial value. The peel had good color, the fruit was sweet and sour, and there was no off-flavor.

[0069] Table 6. Effects of coating agent treatment on the sensory properties of postharvest summer oranges.

[0070] Example 3: Analysis of Functional Thin Film Preparation During post-harvest storage and transportation, citrus fruits suffer from quality decline and high losses due to moisture loss, pathogen infection, and physiological aging, severely hindering industry development. Therefore, polyethylene film bags are commonly used for packaging. While traditional polyethylene film bags offer some barrier properties, they are non-biodegradable and cause white pollution upon disposal, contradicting current green and sustainable development principles. Furthermore, traditional packaging fails to meet the diverse preservation needs of citrus fruits, unable to effectively inhibit pathogen growth or regulate the internal gas environment, leading to a high dependence on preservatives. Against this backdrop, developing new packaging materials that combine excellent preservation performance with environmentally friendly characteristics has become a crucial issue urgently needing to be addressed in the field of post-harvest citrus preservation.

[0071] Building upon Example 2, the use of a linalool-containing coating agent to treat citrus fruits has achieved the effects of reducing rot rates and delaying disease incidence. Against this backdrop, packaging materials need to overcome the limitations of traditional plastics and single-function applications. By developing functional films using biodegradable bio-based materials such as chitosan and pullulan as substrates, and adding linalool to achieve antibacterial properties, these films can achieve preservation while being biodegradable. This reduces the environmental burden on citrus fruits in the post-harvest stage across the entire chain from preservation to packaging materials, promoting the green transformation of the industry.

[0072] 1. Materials and Processing Based on the results of Example 2, functional films containing linalool were prepared and relevant indicators were tested. The specific formulations are shown in Table 7.

[0073] CS: Weigh 1 mL of glacial acetic acid into 99 mL of pure water and stir magnetically to obtain a glacial acetic acid solution. Weigh 1.5 g of chitosan and slowly add it to the glacial acetic acid solution. Stir magnetically for 2 hours under a 70°C water bath until completely transparent to obtain a homogeneous colloidal solution.

[0074] CS / Pu: Weigh 1 mL of glacial acetic acid into 99 mL of pure water and stir magnetically to obtain a glacial acetic acid solution. Weigh 1.5 g of chitosan and slowly add it to the glacial acetic acid solution. Continue stirring magnetically for 2 hours in a 70°C water bath until completely transparent, obtaining a homogeneous colloidal solution. Simultaneously, dissolve 1.5 g of pullulan in 100 mL of pure water and stir in a 45°C water bath for 1 hour until completely dissolved. Mix the two solutions at a volume ratio of 1:1 and continue stirring in a 60°C water bath for 2 hours to obtain the membrane solution used for membrane preparation.

[0075] CS / Pu / L1: Weigh 1 mL of glacial acetic acid into 99 mL of pure water and stir magnetically to obtain a glacial acetic acid solution. Weigh 1.5 g of chitosan and slowly add it to the glacial acetic acid solution. Continue stirring magnetically for 2 hours in a 70°C water bath until completely transparent, obtaining a homogeneous colloidal solution. Simultaneously, dissolve 1.5 g of pullulan in 100 mL of pure water and stir in a 45°C water bath for 1 hour until completely dissolved. Mix the two solutions at a volume ratio of 1:1 and continue stirring in a 60°C water bath for 2 hours. Weigh 0.06 g of linalool and dissolve it in 1 mL of anhydrous ethanol. Slowly add the linalool-ethanol solution dropwise to the homogenized chitosan-pullulan mixture at a dropping rate controlled at 1 mL / min to obtain the membrane solution for preparing the film.

[0076] CS / Pu / L2: Weigh 1 mL of glacial acetic acid into 99 mL of pure water and stir magnetically to obtain a glacial acetic acid solution. Weigh 1.5 g of chitosan and slowly add it to the glacial acetic acid solution. Continue stirring magnetically for 2 hours in a 70°C water bath until completely transparent, obtaining a homogeneous colloidal solution. Simultaneously, dissolve 1.5 g of pullulan in 100 mL of pure water and stir in a 45°C water bath for 1 hour until completely dissolved. Mix 100 mL of each solution and continue stirring in a 60°C water bath for 2 hours to obtain a chitosan-pullulan mixture. Weigh 0.09 g of linalool and dissolve it in 1 mL of anhydrous ethanol. Slowly add the linalool-ethanol solution dropwise to the homogenized chitosan-pullulan mixture at a rate controlled at 1 mL / min to obtain the membrane solution for film preparation.

[0077] CS / Pu / L3: Weigh 1 mL of glacial acetic acid into 99 mL of pure water and stir magnetically to obtain a glacial acetic acid solution. Weigh 1.5 g of chitosan and slowly add it to the glacial acetic acid solution. Continue stirring magnetically for 2 hours in a 70°C water bath until completely transparent, obtaining a homogeneous colloidal solution. Simultaneously, dissolve 1.5 g of pullulan in 100 mL of pure water and stir in a 45°C water bath for 1 hour until completely dissolved. Mix 100 mL of each solution and continue stirring in a 60°C water bath for 2 hours to obtain a chitosan-pullulan mixture. Weigh 0.12 g of linalool and dissolve it in 1 mL of anhydrous ethanol. Slowly add the resulting linalool-ethanol solution dropwise to the homogenized chitosan-pullulan mixture at a rate controlled at 1 mL / min to obtain the membrane solution for film preparation.

[0078] Thin films were prepared using the membrane solutions prepared above: 100 mL of membrane solution was poured into a culture dish with a diameter of about 15 cm, and left to stand in a constant temperature drying oven at 40 °C for 24 hours. The solution was then evaporated to dryness, resulting in a thin film with a diameter of about 15 cm.

[0079] Table 7. Formulation schemes for different functional thin film solutions

[0080] 2. Analysis and Results 2.1 Component analysis of thin films with different ratios Fourier transform infrared spectroscopy is used for omics analysis. For example... Figure 4 As shown, there is good compatibility between the linalool-chitosan-pullulan membrane matrix. Overall, the FTIR spectra of the five samples are highly similar in shape, all exhibiting typical polysaccharide characteristic absorption patterns, indicating that the addition of linalool did not alter the basic chemical structure of the membrane. After the addition of linalool, no obvious signs of phase separation (such as sharp new peaks or severe peak splitting) were observed in the spectra, indicating that linalool can be uniformly dispersed in the membrane matrix, forming a well-compatible composite system. Simultaneously, the formation of interactions such as hydrogen bonds not only contributes to the stable encapsulation of linalool, reducing its volatilization during storage and use, but more importantly, this interaction alters the stacking pattern of the polysaccharide molecular chains, thus potentially significantly improving the membrane's flexibility, mechanical strength, and barrier properties.

[0081] 2.2 Effects of different formulation ratios on the structure of the thin film XRD analysis can obtain information about the membrane structure, such as Figure 5The addition of linalool essential oil altered the regular arrangement of polysaccharide molecular chains in the chitosan-pullulan composite membrane, leading to a decrease in membrane crystallinity and an increase in amorphous regions. The diffraction curves of the linalool-containing samples all became significantly flatter, a characteristic of typical amorphous materials. This change demonstrates that linalool molecules successfully intercalated between the molecular chains of chitosan and pullulan. Linalool transformed the composite membrane from a semi-crystalline state to a highly homogeneous amorphous state, proving its excellent compatibility with the two polysaccharide matrices, promoting molecular-level mixing of the components, and facilitating the formation of a more structurally uniform and structurally stable composite membrane.

[0082] 2.3 Analysis of the effect of different formulation ratios on the mechanical properties of thin films Tensile analysis clearly demonstrates that linalool essential oil effectively improves the mechanical properties of thin films. For example... Figure 6 As shown, the linalool-containing film exhibits characteristics perfectly between those of CS and CS / Pu films, achieving an optimized combination of properties. Its toughness is significantly greater than that of the CS film, while its peak stress (strength) is also significantly higher than that of the CS / Pu film. This indicates that the introduction of linalool simultaneously enhances the material's load-bearing capacity and deformation capacity.

[0083] 2.4 Effect of different formulation ratios on the microstructure analysis of thin films The addition of linalool is a key factor in regulating membrane structure.

[0084] like Figure 7 As shown, the added linalool disrupted the original homogeneous and dense structure, inducing phase separation to form a multi-level structure including particles, pores, and layers inside and on the surface of the membrane. This provides direct morphological evidence for understanding its macroscopic improvement of membrane performance. Pure chitosan films are brittle and have a loose structure with poor surface morphology. The CS / Pu cross-sectional structure is extremely dense and continuous, with no visible pores, and the material is homogeneous, showing strong binding force, indicating that the introduction of pullulan greatly improves film formation. The linalool-containing group exhibits a layered or loose structure, and the CS / Pu / L1 and CS / Pu / L3 groups show more pores, indicating that the addition of linalool interferes with the tight packing of polysaccharide molecules, making the internal structure loose and porous. Although this may be detrimental to barrier properties, the layered structure may help improve toughness.

[0085] 3. Conclusion Linalool is a functional modifier; its addition sacrifices some structural integrity and density in exchange for new functions such as antibacterial and antioxidant properties. While its microstructure is not as good as CS / Pu, it is significantly superior to pure CS film. A composite film of linalool, pullulan, and chitosan possesses mechanical strength, antibacterial properties, and stability, showing promise for citrus preservation. The CS / Pu / L2 group has a relatively dense structure, which is beneficial for the barrier properties of the film bag. Compared to CS / Pu / L1 and CS / Pu / L3, it provides better isolation from external pathogens. Considering barrier properties, film bag mechanical strength, and antibacterial properties, the CS / Pu / L2 group film bag is chosen for citrus preservation.

Claims

1. Application of linalool in any of the following: (1) Use in inhibiting plant pathogens or in preventing and controlling plant diseases caused by said plant pathogens; (2) Use in the preparation of products that inhibit plant pathogens or prevent plant diseases caused by said plant pathogens; The plant pathogens include Penicillium italicum, Penicillium fingernail, or anthracnose.

2. The application according to claim 1, characterized in that: The plant in question is a citrus fruit.

3. The application according to claim 1, characterized in that: The working concentration of linalool is ≥3.2 μL / mL, preferably ≥6.4 μL / mL, and more preferably 3.2~26 μL / mL.

4. A fruit preservative coating agent, characterized in that: It contains the following components: The mixture comprises 0.40-0.80% (v / v) chitosan co-solvent, 1.0-2.0% (w / v) chitosan, 1.0-2.0% (w / v) pullulan, 0.10-0.20% (v / v) linalool, and linalool co-solvent, wherein the mass of the linalool co-solvent is 0.6-1.5% of the mass of linalool, with the balance being water; the chitosan co-solvent is selected from glacial acetic acid, hydrochloric acid, or formic acid, and the linalool co-solvent is selected from methanol, ethanol, or Tween 80; preferably, it contains the following components: 0.40~0.60% (v / v) or 0.45~0.55% (v / v) chitosan co-solvent, 1.3~1.8% (w / v) chitosan, 1.3~1.8% (w / v) pullulan, 0.13~0.18% (v / v) linalool, linalool co-solvent, wherein the mass of the linalool co-solvent is 0.8~1.2% of the mass of linalool; or, 0.5% (v / v) chitosan cosolvent, 1.5% (w / v) chitosan, 1.5% (w / v) pullulan, 0.15% (v / v) linalool, linalool cosolvent, wherein the mass of the linalool cosolvent is 1.0% of the mass of linalool.

5. The method for preparing the fruit preservative coating agent according to claim 4, characterized in that, The process includes the following steps: weigh each component according to the proportion, mix them, stir and mix thoroughly to obtain the final product.

6. The method for preparing the fruit preservative coating agent according to claim 5, characterized in that, The process includes the following steps: adding chitosan as a co-solvent and chitosan to water and stirring thoroughly to dissolve and mix, thus obtaining a chitosan solution; adding pullulan to water and stirring thoroughly to dissolve and mix, thus obtaining a pullulan solution; adding linalool as a co-solvent to linalool and stirring thoroughly; and mixing the prepared chitosan solution, pullulan solution, and linalool containing linalool as a co-solvent, and stirring thoroughly to obtain the final product.

7. An antibacterial film for preserving fruit, characterized in that: The antibacterial film comprises the following components in a mass ratio of 14-18:14-18:1, consisting of chitosan, pullulan, and linalool, preferably in a mass ratio of 15-17:15-17:1 or 16.2-17.2:16.2-17.2:

1.

8. A film-forming solution for an antibacterial film used for fruit preservation, characterized in that: The film-forming solution comprises the following components in the following proportions: 0.40~0.80% (v / v) chitosan co-solvent, 0.5~1.0% (w / v) chitosan, 0.5~1.0% (w / v) pullulan, 0.03~0.06% (w / v) linalool, and 0.40~0.80% (v / v) linalool co-solvent; wherein the chitosan co-solvent is selected from glacial acetic acid, hydrochloric acid, or formic acid, and the linalool co-solvent is selected from methanol, ethanol, or Tween 80; Preferably, the film-forming solution comprises the following components in the following proportions: 0.40~0.60% (v / v) or 0.45~0.55% (v / v) chitosan co-solvent, 0.7~0.8% (w / v) chitosan, 0.7~0.8% (w / v) pullulan, 0.04~0.05% (w / v) linalool, and 0.40~0.60% (v / v) or 0.45~0.55% (v / v) linalool co-solvent; or: 0.50% (v / v) chitosan co-solvent, 0.75% (w / v) chitosan, 0.75% (w / v) pullulan, 0.045% (w / v) linalool, and 0.5% (v / v) linalool co-solvent.

9. The method for preparing the antibacterial film for fruit preservation according to claim 7, characterized in that: Weigh each raw material component according to the proportion, mix and stir evenly, place in a film forming mold, heat to evaporate the solvent, and obtain an antibacterial film; The preferred preparation method includes the following steps: adding chitosan co-solvent and chitosan to water and stirring thoroughly to dissolve and mix, thereby obtaining a chitosan solution; adding pullulan polysaccharide to water and stirring thoroughly to dissolve and mix, thereby obtaining a pullulan polysaccharide solution; adding linalool co-solvent to linalool and mixing thoroughly; mixing the prepared chitosan solution, pullulan polysaccharide solution, and linalool containing linalool co-solvent, and mixing thoroughly to obtain a film-forming solution; placing the film-forming solution in a flat film-forming mold and allowing it to stand at 35~50℃ (preferably 40~45℃) for 20~28h to evaporate the solvent, thereby obtaining an antibacterial film.

10. The application of the fruit preservation coating agent of claim 4, the antibacterial film of claim 7, or the film-forming solution of claim 8 in fruit preservation; The preferred method for applying the fruit preservation coating agent is as follows: fully wet the sponge with the fruit preservation coating agent, and then use the sponge to apply the coating agent to the outer surface of the fruit until the outer surface of the fruit is evenly wetted by the coating agent. The preferred method of applying the antibacterial film is to wrap the fruit in the antibacterial film before storing / transporting it; Preferably, the fruit is a citrus fruit. The coating agent inhibits / kills pathogens carried by the fruit and prevents the fruit from rotting due to pathogens. The pathogens include Penicillium italicum, Penicillium fingernail, or anthracnose.

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