Application of o-vanillin in antibiosis of fruits and vegetables

Abstract

The invention belongs to the technical field of fruit bacteriostasis, and particularly relates to application of o-vanillin in fruit and vegetable antibiosis. The technical problem to be solved by the invention is to screen more plant source antibacterial agents. The technical scheme of the invention is application of o-vanillin in fruit and vegetable antibiosis. The invention provides an effective plant source bacteriostatic agent o-vanillin, which has dual functions of bacteriostasis and resistance induction, not only can inhibit gray mold caused by botrytis cinerea, but also can regulate and control the expression of antioxidant and energy metabolism related genes, and improves the fresh-keeping effect of fruits and vegetables.

Description

Technical Field

[0001] This invention belongs to the field of fruit antibacterial technology, specifically relating to the application of o-vanillin in the antibacterial properties of fruits and vegetables. Background Technology

[0002] Fungal diseases can cause fruit and vegetable spoilage. Gray mold, caused by *Botrytis cinerea*, is one of the major post-harvest diseases. *Botrytis cinerea* typically infects a variety of plant species, including crops, causing significant economic losses. *Botrytis cinerea* infection is a complex process. As a necrotic pathogen, *Botrytis cinerea* enters the host through wounds, stomata, or direct penetration of elliptical conidia. Currently, the application of chemical fungicides is a widely used and effective method for preventing fungal diseases, offering good control, but it is detrimental to food safety and environmental sustainability.

[0003] Plant-derived antibacterial agents are a class of natural chemical substances extracted from plants. They possess significant antibacterial and bacteriostatic activities, effectively inhibiting or killing various bacteria, fungi, and other microorganisms. Compared with traditional chemical antibacterial agents, plant-derived antibacterial agents have the following advantages: (1) High safety, derived from natural plants, with low toxicity to humans and the environment. (2) Broad antibacterial spectrum, effective against various pathogenic and putrefactive bacteria, and less likely to induce drug resistance. (3) Environmentally friendly and sustainable, biodegradable, with minimal impact on the ecological environment. Based on these characteristics, these antibacterial agents have become a research hotspot in the fields of food preservation, agricultural disease control, and medical and health care.

[0004] The main active ingredients of plant-derived antibacterial agents include alkaloids, phenolic compounds, flavonoids, terpenoids, and volatile substances. These components exert their antibacterial effects through several mechanisms, including: disrupting the cell wall and cell membrane structure, leading to nutrient loss and cell death; disrupting the DNA and RNA structure of bacteria, inhibiting gene expression, and causing cell death; disrupting related enzyme systems within the cell, leading to enzyme denaturation and inactivation; and disrupting mitochondrial structure, blocking ATP biosynthesis, inhibiting microbial respiration, and causing cell death due to hypoxia. Most plant-derived antibacterial agents achieve their antibacterial effect by disrupting the bacterial cell wall or cell membrane, altering cell membrane permeability, causing intracellular substances to leak out and bacterial apoptosis. Although there are reports on the application of plant-derived antibacterial agents in postharvest preservation of fruits and vegetables, there are still few reports on safe, efficient, and economically viable plant-derived antibacterial agents. Therefore, screening plant-derived antibacterial agents and elucidating their mechanisms of action has significant theoretical and industrial value. Summary of the Invention

[0005] The technical problem to be solved by this invention is to screen out more plant-derived antibacterial agents.

[0006] The technical solution of this invention is the application of o-vanillin in the antibacterial properties of fruits and vegetables.

[0007] Specifically, the antibacterial agent is effective against Staphylococcus aureus.

[0008] Furthermore, the fruits and vegetables mentioned are apples, berries, or Brassica vegetables.

[0009] Preferably, the apple is a fruit of the genus *Malus*.

[0010] Preferably, the berry fruit is blueberry or strawberry.

[0011] Preferably, the Brassica vegetable is flowering cabbage.

[0012] Another technical solution of the present invention is the application of o-vanillin in the preservation of fruits and vegetables.

[0013] Furthermore, the fruits and vegetables mentioned are apples, berries, or Brassica vegetables.

[0014] Preferably, the apple is a fruit of the genus *Malus*.

[0015] Preferably, the berry fruit is blueberry or strawberry.

[0016] Preferably, the Brassica vegetable is flowering cabbage.

[0017] The present invention also provides a method for improving the resistance of fruits and vegetables to gray mold, wherein the fruits and vegetables are disinfected by spraying or soaking them with o-vanillin solution; wherein the concentration of o-vanillin solution is 2-8 mg / mL.

[0018] Preferably, the concentration of the o-vanillin solution is 2-6 mg / mL.

[0019] Furthermore, the fruits and vegetables mentioned are apples, berries, or Brassica vegetables.

[0020] Preferably, the apple is a fruit of the genus *Malus*.

[0021] Preferably, the berry fruit is blueberry or strawberry.

[0022] Preferably, the Brassica vegetable is flowering cabbage.

[0023] Preferably, the disinfection uses 1-2% NaClO.

[0024] The present invention also provides a method for preserving fruits and vegetables, comprising the following steps: disinfecting the fruits and vegetables, and spraying or soaking the fruits and vegetables with o-vanillin solution; wherein the concentration of o-vanillin solution is 2-8 mg / mL.

[0025] Preferably, the concentration of the o-vanillin solution is 2-6 mg / mL.

[0026] Furthermore, the fruits and vegetables mentioned are apples, berries, or Brassica vegetables.

[0027] Preferably, the apple is a fruit of the genus *Malus*.

[0028] Preferably, the berry fruit is blueberry or strawberry.

[0029] Preferably, the Brassica vegetable is flowering cabbage.

[0030] Preferably, the disinfection uses 1-2% NaClO.

[0031] The beneficial effects of this invention are as follows: This invention provides an effective plant-derived antibacterial agent—o-vanillin. The plant-derived antibacterial agent provided by this invention significantly inhibits the mycelial expansion of *Botrytis cinerea*, and a concentration of 0.2 mg / mL in vitro is sufficient to completely block *Botrytis cinerea* spore germination. Furthermore, o-vanillin can significantly reduce the incidence of gray mold on fruits and vegetables. Further experiments show that o-vanillin achieves its antibacterial effect by disrupting the cell membrane integrity of *Botrytis cinerea*. In addition, o-vanillin can also improve the resistance of fruits and vegetables to gray mold by regulating the expression of resistance-related genes in the metabolic pathways of fruits and vegetables (taking strawberries as an example). o-vanillin improves the preservation effect of fruits and vegetables by regulating the expression of antioxidant and energy metabolism-related genes in fruits and vegetables (taking flowering cabbage as an example). Therefore, this invention discovers that o-vanillin possesses both antibacterial and resistance-inducing functions, providing a theoretical basis for the development of efficient and safe plant-derived preservatives. Its application potential has significant promotional value in the field of postharvest disease control of fruits and vegetables. Attached Figure Description

[0032] Figure 1 The growth status of mycelia on PDA medium under different concentrations of o-vanillin.

[0033] Figure 2 The effect of different concentrations of o-vanillin on the colony growth diameter of Botrytis cinerea on PDA medium and the statistical analysis of its inhibition rate.

[0034] Figure 3 The effect of different concentrations of o-vanillin on the spore germination rate of Botrytis cinerea.

[0035] Figure 4 The effect of different concentrations of o-vanillin on the cell membrane integrity of Botrytis cinerea.

[0036] Figure 5 Treatment with o-vanillin effectively inhibits postharvest yellowing of flowering cabbage.

[0037] Figure 6 Analysis of gene expression levels related to flowering cabbage.

[0038] Figure 7 Treatment with 1,000-1,000 vanillin effectively inhibits postharvest gray mold in strawberries.

[0039] Figure 8 Analysis of the expression levels of genes related to antioxidant and disease resistance in strawberries.

[0040] Figure 9 Ortho-vanillin treatment effectively inhibits postharvest gray mold in blueberries.

[0041] Figure 10 Statistics on the incidence of blueberry disease.

[0042] Figure 11 Ortho-vanillin treatment effectively inhibits postharvest gray mold in apples.

[0043] Figure 12 Statistics on the diameter of pathogens in apples treated with o-vanillin after inoculation with Botrytis cinerea. Detailed Implementation

[0044] o-Vanillin, also known as 2-hydroxy-3-methoxybenzaldehyde or o-vanillin, is a pale yellow needle-like crystal. It is an organic compound with a vanilla bean aroma and a rich, creamy fragrance, belonging to the aromatic aldehyde class. O-Vanillin is an active compound extracted from vanilla orchids, red pine cones, and plants of the *Imperata* genus. It is commonly used as a pharmaceutical intermediate and is an important starting material for the synthesis of various active pharmaceutical ingredients and fragrances, particularly in the synthesis of Schiff bases. It also has wide applications in food, pharmaceuticals, and cosmetics.

[0045] However, there are no reports in the prior art regarding the role of o-vanillin in the antibacterial activity of fruits and vegetables. Therefore, this invention considers exploring the application of o-vanillin in the antibacterial activity of fruits and vegetables.

[0046] First, the in vitro inhibitory effect of o-vanillin on *Botrytis cinerea* was investigated. The results showed that it could inhibit the mycelial growth, spore germination, and germ tube elongation of *Botrytis cinerea*, leading to damage to its cell membrane function. Furthermore, antibacterial experiments were conducted on some fruits and vegetables (apples, strawberries, blueberries, and flowering cabbage). The results showed that o-vanillin could effectively inhibit the growth of *Botrytis cinerea*, thereby significantly reducing disease infection and wilting in fruits and vegetables.

[0047] To further explore the principles behind the above technical effects, this invention further examined the expression of antioxidant and energy metabolism-related genes in fruits and vegetables treated with o-vanillin. The results showed that o-vanillin can regulate the expression of these genes, thereby regulating the signaling pathways to which these genes belong.

[0048] Based on the above experimental results, a series of technical solutions of the present invention were obtained.

[0049] The technical solution of this invention is the application of o-vanillin in the antibacterial properties of fruits and vegetables.

[0050] Specifically, the antibacterial agent is effective against Staphylococcus aureus.

[0051] The fruits and vegetables mentioned are apples, berries, or Brassica vegetables.

[0052] Preferably, the apple is a fruit of the genus *Malus*.

[0053] Preferably, the berry fruit is blueberry or strawberry.

[0054] Preferably, the Brassica vegetable is flowering cabbage.

[0055] Another technical solution of the present invention is the application of o-vanillin in the preservation of fruits and vegetables.

[0056] The fruits and vegetables mentioned are apples, berries, or Brassica vegetables.

[0057] Preferably, the apple is a fruit of the genus *Malus*.

[0058] Preferably, the berry fruit is blueberry or strawberry.

[0059] Preferably, the Brassica vegetable is flowering cabbage.

[0060] The present invention also provides a method for improving the resistance of fruits and vegetables to gray mold, comprising the following steps: disinfecting the fruits and vegetables, and spraying or soaking the fruits and vegetables with o-vanillin solution; wherein the concentration of o-vanillin solution is 2-8 mg / mL.

[0061] Preferably, the concentration of the o-vanillin solution is 2-6 mg / mL.

[0062] The fruits and vegetables mentioned are apples, berries, or Brassica vegetables.

[0063] Preferably, the apple is a fruit of the genus *Malus*.

[0064] Preferably, the berry fruit is blueberry or strawberry.

[0065] Preferably, the Brassica vegetable is flowering cabbage.

[0066] Preferably, the disinfection uses 1-2% NaClO.

[0067] The present invention also provides a method for preserving fruits and vegetables, comprising the following steps: disinfecting the fruits and vegetables, and spraying or soaking the fruits and vegetables with o-vanillin solution; wherein the concentration of o-vanillin solution is 2-8 mg / mL.

[0068] Preferably, the concentration of the o-vanillin solution is 2-6 mg / mL.

[0069] The fruits and vegetables mentioned are apples, berries, or Brassica vegetables.

[0070] Preferably, the apple is a fruit of the genus *Malus*.

[0071] Preferably, the berry fruit is blueberry or strawberry.

[0072] Preferably, the Brassica vegetable is flowering cabbage.

[0073] Preferably, the disinfection uses 1-2% NaClO.

[0074] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are merely illustrative of the invention and not intended to limit its scope. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the materials, reagents, instruments, etc., used are all commercially available.

[0075] Example 1: Effect of o-vanillin on the mycelial growth of Botrytis cinerea

[0076] The concentration to be prepared is 2×10 6 Botrytis cinerea spore suspension at spores / mL was prepared, followed by dissolution of o-vanillin and serial dilution, and mixed with PDA medium. The mixture was then poured into plates to achieve concentrations of 0, 0.05, 0.1, 0.15, 0.2, and 0.3 mg / mL. 5 μL of spore suspension was inoculated into the center of each plate. Each group was divided into 3 replicates and repeated twice. The plates were incubated upside down at 25 °C for 6 consecutive days to observe mycelial growth.

[0077] The inhibition rate was calculated using the following formula: Growth inhibition rate = (Control colony diameter - Treated colony diameter) / Control colony diameter × 100%

[0078] The addition of o-vanillin to the culture medium showed a concentration-dependent inhibitory effect, with the inhibitory effect increasing with increasing o-vanillin concentration. Figure 1 As shown, during the inoculation and culture process, the lesions decreased in size with increasing o-vanillin concentration. Specifically, when the o-vanillin concentration was 0.30 mg / mL, no significant colony growth was observed after 6 days of culture. Further analysis of the bacterial diameter and calculation of the growth inhibition rate were performed. Figure 2 As shown, after 2 days of inoculation and culture, 0.10 mg / mL o-vanillin inhibited Botrytis cinerea by 63.70%, while 0.15 mg / mL o-vanillin completely inhibited its growth. After 5 days of inoculation and culture, 0.20 mg / mL o-vanillin inhibited Botrytis cinerea by 74.68%, while no trace of Botrytis cinerea growth was observed in the 0.30 mg / mL o-vanillin treatment group. This indicates that with increasing o-vanillin concentration, the colony diameter significantly decreased, demonstrating a significant inhibitory effect on the mycelial spread of Botrytis cinerea.

[0079] Example 2: Determination of spore germination rate of *Botrytis cinerea* after treatment with o-vanillin

[0080] In a clean bench, a sample of o-vanillin stock solution was taken and thoroughly mixed with PDB medium to achieve final concentrations of 0, 0.05, 0.1, and 0.15 mg / mL, with a total volume of 1 mL. The prepared concentration was 2 × 10⁻⁶ mg / mL. 8 A conidial suspension of spores / mL was used, with 10 μL of the suspension inoculated into PDB media of varying concentrations as described above, and incubated at 25 ℃. Samples were taken at 6, 9, and 12 h post-inoculation for observation. After mixing the cultures from each group, 20 μL of the liquid was taken for spore germination observation under an optical microscope, with germ tube length measured using an eyepiece micrometer. Germinated spores were defined as those with a germ tube length greater than their diameter, and the spore germination rate was defined as the percentage of germinating spores out of the total number of spores. Three fields of view were randomly selected, with approximately 200 spores observed each time. Three biological replicates were performed.

[0081] like Figure 3 As shown, the germination rate of *Botrytis cinerea* spores in the control group was 71.06% after 6 h of culture, while the germination rate in the medium with o-vanillin concentration of 0.05 mg / mL was only 12.89%. After 12 h of culture, the germination rate of spores in the control group reached as high as 99.72%, while the germination rate of spores in the medium with o-vanillin concentration of 0.05 mg / mL was 50.44%, and the spores in the medium with o-vanillin concentrations of 0.1 mg / mL and above still failed to germinate.

[0082] Example 3: PI staining to detect the effect of o-vanillin on the cell membrane integrity of *Botrytis cinerea*.

[0083] Propidium iodide (PI) cannot penetrate intact cell membranes, but it can penetrate damaged cell membranes of dead cells. The effect of o-vanillin on the cell membrane integrity of *Botrytis cinerea* can be detected using PI staining solution. In a clean bench, o-vanillin stock solution was aspirated and thoroughly mixed with PDB medium. Final concentrations were set at 0, 0.05, 0.1, 0.15, 0.2, and 0.3 mg / mL for six treatment groups, with a total volume of 4 mL. A concentration of 2 × 10⁻⁶ mg / mL was prepared. 6A conidial suspension with spores / mL was inoculated into different concentrations of PDB medium, and cultured at 28℃ and 200 rpm for 6 h in a constant temperature shaker. The culture was centrifuged at 9800 rpm for 2 min at room temperature to obtain a precipitate. The precipitate was washed three times with PBS and then resuspended in 500 μL of PBS. 500 μL of PI staining solution with a concentration of 200 μg / mL was added to bring the final PI staining solution concentration to 100 μg / mL. After thorough mixing, the stained sample was placed in a constant temperature shaker incubator at 28℃ and 200 rpm for 30 min in the dark. After staining, the precipitate was washed once with PBS and observed under a fluorescence microscope. Three fields of view were randomly selected for photographing, and the results were observed. Figure 4 As the concentration of o-vanillin increased, the intensity of the corresponding red fluorescence significantly increased. This indicates that o-vanillin can disrupt cell membrane integrity in vitro, and the degree of disruption increases with increasing o-vanillin concentration. These results suggest that o-vanillin inhibits spore germination by disrupting cell membrane integrity.

[0084] Example 4: Ortho-vanillin treatment effectively delays postharvest leaf yellowing in flowering cabbage.

[0085] After disinfection and cleaning, flowering stalks (scientific name: *Brassica rapa* var. *chinensis* 'Parachinensis') were sprayed with 5 mL of o-vanillin solution at concentrations of 0, 2, 4, and 6 mg / mL, respectively, and then air-dried. The treated flowering stalks were stored at 25 °C, and yellowing and rotting were observed every 24 hours to evaluate the effect of different concentrations of o-vanillin on postharvest preservation. Simultaneously, the expression levels of AOX2 and CHI in the flowering stalks were analyzed; the primer sequences used are shown in Table 1.

[0086] Observations showed that spraying with 6 mg / mL o-vanillin effectively delayed the yellowing of flowering cabbage leaves and slowed down the rate of tissue decay. For example... Figure 5 On day 3 after treatment, the leaves of the control group and the low-concentration o-vanillin spray treatment group showed yellowing and the stem tissue showed rotting. No obvious yellowing or rotting was found in the 6 mg / mL o-vanillin spray treatment group. On day 6, the leaves of the control group and the low-concentration o-vanillin spray treatment group showed obvious yellowing and the stem tissue showed obvious rotting. However, the leaves of the 6 mg / mL o-vanillin spray treatment group showed a significantly lower degree of yellowing and no rotting was found.

[0087] Postharvest metabolic activity in flowering cabbage is high, leading to rapid leaf senescence and resulting in quality deterioration such as yellowing, wilting, and nutrient loss. Energy status is a key factor in plant senescence; energy supply in plants is jointly regulated by the activity of a series of energy regulatory elements, transcriptional processes, and translational processes, among which dissipative proteins (alternative oxidases, AOX) are one of the main regulatory factors. Figure 6 a. During the storage of flowering cabbage, the expression level of BrAOX2 was significantly reduced after treatment with o-vanillin compared to the control group, which was approximately 20.73 and 29.4 times higher than that of the 4 mg / mL and 6 mg / mL o-vanillin treatment groups, respectively. Normal expression of the BrCHI gene is a key factor in preventing yellowing of flowering cabbage leaves. BrCHI encodes a chalcone isomerase, which converts chalcones into flavanones, thereby reducing chalcone accumulation by promoting flavonoid synthesis, maintaining chloroplast antioxidant capacity, and inhibiting chlorophyll degradation. Figure 6 b. During the storage of flowering cabbage, the expression level of BrCHI in the control group and the low-concentration o-vanillin treatment group gradually decreased, while the expression level of BrCHI in the high-concentration o-vanillin treatment group showed less change, remaining at a certain level and significantly higher than that in the control group. By day 6, the expression levels of the BrCHI gene in the 4 mg / mL and 6 mg / mL o-vanillin treatment groups were approximately 2.47 times and 1.79 times that of the control group, respectively. Treatment with o-vanillin delayed the yellowing of flowering cabbage by regulating the expression of BrCHI and BrAOX2 in the energy metabolism and antioxidant pathways of flowering cabbage, thus improving the preservation effect.

[0088] Example 5: Ortho-vanillin treatment effectively inhibits postharvest gray mold in strawberries.

[0089] The strawberries were disinfected and washed with 1% NaClO for 2 min to remove microorganisms from the fruit surface, then rinsed three times with sterile water and air-dried. They were then soaked in o-vanillin solutions with concentrations of 0, 2, 4, and 6 mg / mL for 10 min each and air-dried for 2–3 h to allow for full absorption of the o-vanillin. Holes were then punched at the equator using a sterile punch, with a diameter of approximately 2 mm and a depth of approximately 2 mm. 5 μL of a 2×10⁻⁶ solution was added to each hole. 6 A suspension of *Botrytis cinerea* spores at spore counts / mL was stored at 25°C, and strawberry rot was observed every 24 hours. Simultaneously, the expression levels of Cu / Zn SOD, POD12, PPO, and CHI in strawberries were detected. The primer sequences used are shown in Table 1.

[0090] Table 1 qRT-PCR primers

[0091] .

[0092] The effects of different concentration gradients of o-vanillin on the incidence and rot of postharvest gray mold in strawberries, such as Figure 7After treatment with 6 mg / mL o-vanillin, no obvious disease was observed on day 2 of storage. The strawberry fruits were more vibrant and red in color compared to the control group, indicating a better inhibitory effect against *Botrytis cinerea*. By day 4, the mycelial growth in the control group was extensive, covering the entire strawberry surface, while the 6 mg / mL o-vanillin treatment group showed less mycelial spread and milder disease compared to other treatment groups and the control group. O-vanillin significantly inhibited the rot caused by *Botrytis cinerea* infection in strawberries, effectively delaying fruit decay.

[0093] Superoxide dismutase (SOD) catalyzes the conversion of superoxide radicals (O2⁻) into hydrogen peroxide and oxygen, relying on copper (Cu) and zinc (Zn) as cofactors. It forms the first line of defense in the antioxidant system, and o-vanillin treatment upregulates FaCu / ZnSOD expression. Figure 8 a. In the control group, the expression level of FaCu / ZnSOD in strawberries was high on day 2, but significantly decreased on day 4. In the o-vanillin treatment group, the relative expression of FaCu / ZnSOD gradually increased during storage, while the expression level in the high-concentration (6 mg / mL) o-vanillin treatment group was significantly increased, approximately 1.46 times that of the control group. Peroxidase (POD) can decompose reactive oxygen species such as hydrogen peroxide and participate in disease resistance and oxidative stress response. O-vanillin treatment can upregulate FaPOD12 expression. Figure 8 b. In strawberries treated with o-vanillin, the expression of FaPOD12 significantly increased on day 4 after o-vanillin treatment. The gene expression level after o-vanillin treatment was significantly higher than that in the control group, with the 4 mg / mL and 6 mg / mL o-vanillin treatment groups being approximately 1.80 times and 4.29 times higher than the control group, respectively. Polyphenol oxidase (PPO) catalyzes the oxidation of polyphenols to quinones, participating in plant disease resistance responses. Its gene expression plays an important role in postharvest disease prevention in fruits and vegetables. After *Botrytis cinerea* infection of strawberries, the expression of the FaPPO gene gradually increased during strawberry storage, and its expression level was positively correlated with the o-vanillin treatment concentration. Figure 8 c. On day 4, the FaPPO gene expression level in the 6 mg / mL o-vanillin treatment group was approximately 2.01 times that of the control group. Chitinase (CHI) hydrolyzes chitin in the fungal cell wall, directly inhibiting fungal growth. The released chitin oligosaccharides can act as elicitors to trigger plant immune responses. O-vanillin treatment significantly increased its gene expression, such as... Figure 8Two days after *Botrytis cinerea* infection of strawberries, the expression of the FaCHI gene in both the control group and the low-concentration o-vanillin (4 mg / mL) treatment group decreased relatively. By day 4, the control group had not recovered to its initial level, while the expression level in the 4 mg / mL o-vanillin treatment group significantly increased. At day 4, the expression levels in the 4 mg / mL and 6 mg / mL o-vanillin treatment groups were approximately 3.81 times and 4.66 times that of the control group, respectively. These results indicate that o-vanillin treatment of strawberries can induce the upregulation of antioxidant enzyme-related genes Cu / ZnSOD, POD12, and PPO, as well as the disease resistance-related gene CHI, thereby enhancing the fruit's disease resistance.

[0094] Example 6: Ortho-vanillin treatment effectively inhibits postharvest gray mold in blueberries

[0095] Select fresh blueberries, sterilize with 1% NaClO for 2 minutes, rinse with sterile water and air dry. Use a sterile punch to make holes (2 mm × 2 mm) at the stem end of the blueberries. Prepare 2 × 10 6 Botrytis cinerea spore suspension (spores / mL) was inoculated into each well with 5 μL of spore suspension. After 12 h, 5 μL of o-vanillin at a gradient concentration was added to each well. The wells were incubated at 25 ℃, and disease incidence was observed and recorded every 24 h. Figure 9 Four days after inoculation, the blueberries in the control group were covered by mycelial spread, while the o-vanillin treatment significantly inhibited the occurrence of gray mold, and its inhibitory effect increased with increasing o-vanillin concentration. For example, two days after inoculation, the lesion diameter in the 6 mg / mL and 8 mg / mL o-vanillin treatment groups was significantly reduced compared to the control group, at 65.52% and 4.54% of the control group, respectively; four days after inoculation, the incidence rate in the control group was approximately 100%, while the incidence rate in the 8 mg / mL o-vanillin treatment group was significantly reduced, only 56.57% of the control group. Figure 10 ).

[0096] Example 7: Ortho-vanillin treatment significantly inhibited postharvest gray mold in apples.

[0097] Select healthy, undamaged Red Dew apples. Disinfect with 1% NaClO for 2 minutes, then wash with sterile water and air dry. Use a sterile punch to make a 5 mm × 5 mm hole at the equator of each apple, and add 20 μL of a pre-prepared solution with a concentration of 2 × 10⁻⁶. 6 A suspension of *Botrytis cinerea* spores (spores / mL) was prepared, and after the liquid was aspirated, 20 μl of o-vanillin solution at concentrations of 0, 2.5, 5, and 10 mg / mL was added. The mixture was incubated at 25 °C, and after 3 days, disease development was observed and the diameter of the pathogens was counted. Figure 11The diameter of the pathogen increased with the number of incubation days. The diameter of the decaying apples treated with o-vanillin was significantly smaller than that of the control group. During days 3-6 of incubation, all concentrations of o-vanillin effectively inhibited the expansion of the pathogen diameter, with the 2 mg / mL o-vanillin treatment group showing the best results. The pathogen diameters at 3-6 days were 41.53%, 48.35%, 61.05%, and 72.02% of the control group, respectively. Figure 12 ).

Claims

1. Application of o-vanillin in antibacterial properties of fruits and vegetables.

2. The application according to claim 1, characterized in that: The antibacterial agent is effective against Staphylococcus aureus.

3. The application according to claim 1, characterized in that: The fruits and vegetables are apples, berries, or Brassica vegetables; preferably, the apples are apples; preferably, the berries are blueberries or strawberries; preferably, the Brassica vegetables are flowering cabbage.

4. Application of o-vanillin in fruit and vegetable preservation.

5. The application according to claim 4, characterized in that: The fruits and vegetables are apples, berries, or Brassica vegetables; preferably, the apples are apples; preferably, the berries are blueberries or strawberries; preferably, the Brassica vegetables are flowering cabbage.

6. A method for improving the resistance of fruits and vegetables to gray mold, characterized in that: The procedure includes the following steps: disinfecting fruits and vegetables by spraying or soaking them with an o-vanillin solution; wherein the concentration of the o-vanillin solution is 2-8 mg / mL; preferably, the concentration of the o-vanillin solution is 2-6 mg / mL.

7. A method for preserving fruits and vegetables, characterized in that: The procedure includes the following steps: disinfecting fruits and vegetables by spraying or soaking them with an o-vanillin solution; wherein the concentration of the o-vanillin solution is 2-8 mg / mL; preferably, the concentration of the o-vanillin solution is 2-6 mg / mL.

8. The method according to claim 6 or 7, characterized in that: The fruits and vegetables are apples, berries, or Brassica vegetables; preferably, the apples are apples; preferably, the berries are blueberries or strawberries; preferably, the Brassica vegetables are flowering cabbage.

9. The method according to claim 6 or 7, characterized in that: The disinfection process uses 1-2% NaClO.

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