A method for improving the preservation ability of peppers based on a chitosan / nitrogen-doped carbon dot composite system

Abstract

This invention discloses a method for improving the preservation ability of chili peppers based on a chitosan / nitrogen-doped carbon dot composite system, belonging to the field of novel pesticides. The application of chitosan / nitrogen-doped carbon dots in improving the preservation ability of chili peppers involves preparing chitosan / nitrogen-doped carbon dots into a suspension or a film, and then applying it to chili pepper fruits through impregnation, spraying, and film packaging. The chitosan / nitrogen-doped carbon dot film exhibits excellent antibacterial and antioxidant capabilities, good ultraviolet shielding ability, and water, oxygen, and carbon dioxide barrier capabilities. This invention improves the preservation ability of chili peppers through post-harvest chitosan / nitrogen-doped carbon dot impregnation, spraying, or film packaging.

Description

Technical Field

[0001] This invention relates to a method for improving the preservation ability of chili peppers based on a chitosan / nitrogen-doped carbon dot composite system, which belongs to the field of novel pesticides. Background Technology

[0002] Globally, approximately 1.3 billion tons of food intended for human consumption are wasted annually, with vegetables and fruits accounting for 33%. Besides causing enormous economic losses, spoiled fruits and vegetables also produce numerous toxic secondary metabolites, such as various mycotoxins and allergens. These substances have potential carcinogenic and mutagenic properties and negatively impact human health. Existing solutions to these losses include refrigeration, the addition of preservatives, waxing treatments, and controlled atmosphere packaging. However, these methods suffer from drawbacks such as high costs or safety concerns. Therefore, there is an urgent need for technologies that preserve perishable fruits and vegetables in a convenient, effective, and sustainable manner. Summary of the Invention

[0003] [Technical Issues]

[0004] This provides a convenient, effective, and sustainable new method for preserving perishable fruits and vegetables.

[0005] [Technical Solution]

[0006] To address the aforementioned issues, this invention prepares CS / N-CDs into a suspension or a thin film, which is then applied to chili pepper fruits for storage, thereby enhancing the preservation capabilities of the chili pepper fruits.

[0007] The first objective of this invention is to provide the application of a chitosan / nitrogen-doped carbon dot composite system (CS / N-CDs) in enhancing the preservation ability of chili peppers.

[0008] The preparation method of the CS / N-CDs composite system includes:

[0009] Carbon and nitrogen sources were dispersed in water for a hydrothermal reaction. After the reaction was completed, the mixture was cooled, filtered through a membrane, and freeze-dried to obtain nitrogen-doped carbon dots (denoted as N-CDs). Chitosan was dispersed in an acetic acid solution, then glycerol was added and mixed. The obtained N-CDs were then added and stirred to react, resulting in a chitosan / nitrogen-doped carbon dot composite system, denoted as CS / N-CDs.

[0010] In one embodiment of the invention, the application involves immersing or spraying CS / N-CDs in the form of a suspension onto chili pepper fruits.

[0011] In one embodiment of the invention, the application involves wrapping CS / N-CDs in the form of a film onto pepper fruits.

[0012] In one embodiment of the present invention, the soaking involves immersing the fruit in a CS / N-CDs suspension, wiping it after soaking, air-drying it, and storing it at room temperature.

[0013] In one embodiment of the present invention, the spraying involves spraying a CS / N-CDs suspension onto chili peppers (calculated to yield 1.25 mL of spray solution per chili pepper), air-drying, and then storing at room temperature.

[0014] In one embodiment of the present invention, the CS / N-CDs film is obtained by spreading a CS / N-CDs suspension (i.e., a chitosan / nitrogen-doped carbon dot composite system), then letting it stand and drying it for a period of time.

[0015] In one embodiment of the present invention, the CS / N-CDs suspension is a 0.5% CS / N-CDs suspension (N-CDs:CS = 0.5%, that is, the mass ratio of N-CDs to CS is 0.005:1).

[0016] In one embodiment of the present invention, the chili pepper is a bird's eye chili.

[0017] In one embodiment of the present invention, in the preparation method of the CS / N-CDs composite system, the carbon source is citric acid or its hydrate.

[0018] In one embodiment of the present invention, the nitrogen source in the preparation method of the CS / N-CDs composite system is urea.

[0019] In one embodiment of the present invention, in the preparation method of the CS / N-CDs composite system, the mass ratio of carbon source to nitrogen source is 1:1.5.

[0020] In one embodiment of the present invention, in the preparation method of the CS / N-CDs composite system, the carbon source has a dispersion concentration of 0.1 g / mL in water.

[0021] In one embodiment of the present invention, the hydrothermal reaction conditions in the preparation method of the CS / N-CDs composite system are 150-200℃ for 3-8 hours. Specifically, the reaction can be carried out at 180℃ for 6 hours.

[0022] In one embodiment of the present invention, in the preparation method of the CS / N-CDs composite system, a 0.22 μm polyethersulfone (PES) membrane is used for membrane filtration.

[0023] In one embodiment of the present invention, in the preparation method of the CS / N-CDs composite system, the dispersion concentration of chitosan in the chitosan-acetic acid solution is 0.02 g / mL.

[0024] In one embodiment of the present invention, in the preparation method of the CS / N-CDs composite system, the acetic acid solution is an aqueous solution of acetic acid with a volume fraction of 1%.

[0025] In one embodiment of the present invention, in the preparation method of the CS / N-CDs composite system, the volume fraction of glycerol relative to the acetic acid solution is 2%.

[0026] In one embodiment of the present invention, in the preparation method of the CS / N-CDs composite system, the mass ratio of N-CDs to chitosan is (0.001-0.025):1; further optionally, it is 0.005:1.

[0027] In one embodiment of the present invention, the preparation method of the CS / N-CDs composite system (CS / N-CDs suspension) specifically includes the following steps:

[0028] Synthesis steps of N-CDs: Weigh 4.0 g of citric acid monohydrate and 6.0 g of urea, add 40 mL of ultrapure water, then transfer the mixed solution to a reaction vessel (100 mL), react at 180 °C for 6 h, cool at room temperature, filter the resulting suspension using a 0.22 μm polyethersulfone (PES) membrane to remove large particles, wash with ultrapure water and freeze-dry to obtain N-CDs.

[0029] Synthesis steps of CS / N-CDs suspension and film: Nanocomposite films were prepared by solvent casting using CS and N-CDs as raw materials. 2.0 g of chitosan was weighed and added to 100 mL of 1 vol% acetic acid solution, stirred at room temperature for 3 h, followed by the addition of 2 vol% glycerol, and stirring again for 4 h. Then, 10 mg of N-CDs particles were added, and the mixture was sonicated for 1 h to obtain a CS / N-CDs mixture, designated as a 0.5% CS / N-CDs suspension.

[0030] The second objective of this invention is a method for reducing the weight loss of chili peppers during storage using CS / N-CDs. The method involves preparing CS / N-CDs into a CS / N-CDs suspension and then applying it to the chili peppers by soaking or spraying.

[0031] The third objective of the invention is a preservation method for regulating the distribution of microorganisms in chili pepper fruits. This method utilizes CS / N-CDs to downregulate the relative abundance of microorganisms associated with rotting and shriveling of chili pepper fruits and to upregulate the relative abundance of beneficial microorganisms. The method involves preparing CS / N-CDs into a CS / N-CDs suspension and then applying it to chili pepper fruits by soaking or spraying.

[0032] In one embodiment of the invention, the microorganisms associated with fruit rot and shriveling include Enterobacter, Chryseobacterium, and Ralstonia; the beneficial microorganisms include Pseudomonas, Arthrobacter, Sphingobacterium, Paenibacillus, and Ochrobactrum.

[0033] In one embodiment of the present invention, the preservation ability of chili peppers is improved by lowering the relative abundance of microorganisms associated with the decay and shriveling of chili peppers and increasing the relative abundance of beneficial microorganisms.

[0034] In one embodiment of the present invention, CS / N-CDs suspension impregnation involves wiping the fruit after soaking and then air-drying it, and storing it at room temperature; CS / N-CDs suspension spraying involves spraying the suspension onto the chili pepper fruit (calculated to yield 1.25 mL of spray solution per chili pepper), air-drying it, and then storing it at room temperature.

[0035] In one embodiment of the present invention, the CS / N-CDs suspension is a 0.5% CS / N-CDs suspension (N-CDs:CS = 0.5%).

[0036] The third objective of this invention is a method for improving the preservation of chili peppers by delaying the loss of fruit quality and the decrease in firmness, delaying the transpiration and respiration of chili pepper fruits, thereby delaying the decay and deterioration of chili peppers. The method involves preparing CS / N-CDs into CS / N-CDs films and then packaging them onto chili pepper fruits.

[0037] In one embodiment of the present invention, the CS / N-CDs film is a 0.5% CS / N-CDs film (N-CDs:CS = 0.5%).

[0038] In one embodiment of the present invention, the CS / N-CDs film is obtained by spreading a CS / N-CDs suspension, then letting it stand and drying it for a period of time.

[0039] In one embodiment of the present invention, the CS / N-CDs film is obtained by pouring a CS / N-CDs suspension into a petri dish (length × width × height: 12cm × 12cm × 2cm) and placing it in an oven at 60°C for 6 hours.

[0040] [Beneficial Effects]

[0041] (1) This invention improves the preservation ability of chili peppers through CS / N-CDs suspension. Immersion or spraying with 0.5% CS / N-CDs suspension reduces the weight loss of chili peppers during storage (weight loss rate of CS / N-CDs suspension immersion or spraying treatment decreased by 8.7-15.3% compared with the control group); CS / N-CDs suspension immersion or spraying treatment down-increases the relative abundance of microorganisms (Enterobacter, Chryseobacterium and Ralstonia) related to fruit rot and shriveling (65.0-98.2%), while up-increases the relative abundance of beneficial microorganisms (Pseudomonas, Arthrobacter, Sphingobacterium, Paenibacillus and Ochrobactrum) (61.4-99.9%), providing a favorable micro-ecological environment for chili pepper storage.

[0042] (2) The CS / N-CDs film prepared by the present invention exhibits excellent antibacterial and antioxidant capabilities, good ultraviolet shielding capabilities, and water, oxygen and carbon dioxide barrier capabilities.

[0043] (3) This invention improves the preservation ability of chili peppers through CS / N-CDs film. Treatment with 0.5% CS / N-CDs film delays the decay of chili peppers during storage, delays the loss of fruit weight and decrease in firmness (CS / N-CDs film reduced weight loss by 17.3% and increased firmness by 100.0% compared with the control group). Attached Figure Description

[0044] Figure 1 FTIR spectra and barrier and antioxidant properties of CS / N-CDs films. (A) FTIR spectrum of the film; (B) UV-Vis absorption spectrum of the film; (C) Water vapor permeability; (D) Oxygen permeability; (E) Carbon dioxide permeability; (F) DPPH- free radical scavenging activity. Different letters in the bar chart indicate significant differences between groups (p<0.05).

[0045] Figure 2 The antimicrobial properties of CS / N-CDs films were evaluated. (A) Effects of the films on the growth of *Escherichia coli* and *Staphylococcus aureus* under darkness and ultraviolet light; (B) Relative inhibition rate of the films against *Escherichia coli*; (C) Relative inhibition rate of the films against *Staphylococcus aureus*. Different letters in the bar chart indicate significant differences between groups (p<0.05).

[0046] Figure 3 Effects of CS / N-CDs suspension immersion on (left) phenotype and (right) weight loss of pepper fruits during storage.

[0047] Figure 4The changes in microorganisms in pepper fruits after immersion in CS / N-CDs suspension during storage. (A) Relative abundance of dominant bacterial communities at the phylum level; (B) Relative abundance of the main genera of dominant bacterial communities. Different letters in the figure indicate significant differences between groups (p<0.05).

[0048] Figure 5 The effects of spraying CS / N-CDs suspension on (A) phenotype and (B) weight loss of pepper fruits during storage.

[0049] Figure 6 The changes in microorganisms during the storage of pepper fruits after spraying with CS / N-CDs suspension. (A) Relative abundance of dominant bacterial communities at the phylum level; (B) Relative abundance of the main genera of dominant bacterial communities. Different letters in the figure indicate significant differences between groups (p<0.05).

[0050] Figure 7 The effects of CS / N-CDs film on (A) phenotype, (B) weight loss, and (C) firmness of pepper fruits during storage. Different letters in the bar chart indicate significant differences between groups (p<0.05). Detailed Implementation

[0051] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0052] Test method:

[0053] 1. FTIR test:

[0054] The functional groups of the thin film were analyzed using FTIR.

[0055] 2. Absorption spectroscopy test of CS / N-CDs thin films:

[0056] The functional groups of the thin film were analyzed using FTIR, and the absorption spectrum of the thin film was measured in the wavelength range of 200-800 nm using a UV-Vis spectrophotometer to evaluate the light blocking performance of the thin film.

[0057] 3. Barrier performance test:

[0058] The barrier properties of the membrane samples against water vapor, oxygen, and carbon dioxide were evaluated by measuring water vapor permeability (WVP), oxygen permeability (OP), and carbon dioxide permeability (CDP) separately. Centrifuge tubes containing 5 g of anhydrous calcium chloride, an oxygen absorber, and potassium hydroxide were sealed with the membrane samples, and the initial weight was recorded. The tubes were then stored in an incubator (temperature: 25°C, relative humidity: 75%), and the weight of the membrane-covered centrifuge tubes was recorded after 48 hours. WVP, OP, and CDP were calculated using the following formulas:

[0059]

[0060]

[0061]

[0062] Where W represents the increase in weight of the centrifuge tube (g); d represents the thickness of the membrane (m); and A represents the permeation area of ​​the membrane (m²). 2 ); t represents the storage time (s).

[0063] 4. Antioxidant performance test:

[0064] The antioxidant properties of the thin film samples were reflected by measuring the free radical scavenging capacity (DPPH). The detailed procedure is as follows: 50 mg of CS / N-CDs thin film samples with different N-CDs doping ratios (0%, 0.1%, 0.5%, and 2.5%) were dissolved in 1 mL of 1% acetic acid, then transferred to a 10 mL centrifuge tube. 3 mL of DPPH solution was added, and the reaction was carried out at room temperature in the dark for 30 min. Anhydrous ethanol was used as a blank, and the absorbance was measured at 517 nm.

[133] The DPPH free radical scavenging rate is calculated using the following formula:

[0065]

[0066] Where A0 represents the absorbance values ​​of distilled water and DPPH solution, A1 represents the absorbance values ​​of sample solution and DPPH solution, and A2 represents the absorbance values ​​of sample solution and anhydrous ethanol.

[0067] 5. Antibacterial performance test:

[0068] The antibacterial properties of the film samples were evaluated by measuring their antibacterial activity against Gram-negative bacteria (Escherichia coli, for example) and Gram-positive bacteria (Staphylococcus aureus, for example).

[134] The detailed steps are as follows: First, add 0.5 mL of bacterial solution (~10 mL) 7 CFU mL -1CS / N-CDs film samples (20 mm × 20 mm) with different N-CDs incorporation ratios (0%, 0.1%, 0.5%, and 2.5%) were mixed in sterile centrifuge tubes (10 mL) and then placed in a shaker (37°C, 190 rpm). The mixture was divided into two groups: one group was treated under UV irradiation, and the other group was treated in the dark. After 1 h, 150 μL of the treated bacterial solution was evenly spread on a petri dish containing LB medium and incubated for 16 h. Colonies were then counted. The control group underwent similar treatment to the experimental group, except that no film sample was added to the bacterial solution. Finally, the relative inhibition rate was calculated using the following formula:

[0069]

[0070] Where N0 represents the number of colonies in the blank group and N1 represents the number of colonies in the experimental group.

[0071] 6. Testing of fruit weight loss rate and firmness

[0072] The weight loss rate was determined by weighing. The initial weight of the fruit was recorded as M0, and the weight at the sampling point was recorded as M1. The weight loss rate was calculated using the following formula:

[0073]

[0074] The firmness of pepper fruits during storage was determined using a texture analyzer. Specific parameters were: puncture speed: 0.5 mm / s; puncture depth: 7 mm. Fruit firmness was expressed in N.

[0075] 7. Rhizosphere microbial testing:

[0076] On the 21st day after harvesting, the pepper fruits were stored in liquid nitrogen and then sent to Shenzhen MicroMed Technology Group Co., Ltd. for sequencing analysis. First, DNA was extracted and detected. Then, PCR amplification of the V5-V7 region of bacterial 16S rRNA was performed using forward primer 799F (AACMGGATTAGATACCCKG) and reverse primer 1193R (ACGTCATCCCCACCTTCC). After product purification, library preparation, and library testing, sequencing was performed on the Illumina platform.

[0077] Example 1

[0078] A method for preparing CS / N-CDs suspension and thin film includes the following steps:

[0079] Synthesis steps of nitrogen-doped carbon dots: Weigh 4.0 g of citric acid monohydrate and 6.0 g of urea, add 40 mL of ultrapure water, then transfer the mixed solution to a reaction vessel (100 mL), react at 180 °C for 6 h, cool at room temperature, filter the resulting suspension using a 0.22 μm polyethersulfone (PES) membrane to remove large particles, wash with ultrapure water and freeze dry to obtain N-CDs.

[0080] Synthesis steps of CS / N-CDs suspension and film: Nanocomposite films were prepared by solvent casting using CS and N-CDs as raw materials. 2.0 g of CS was weighed and added to 100 mL of 1 vol% acetic acid solution, stirred at room temperature for 3 h, followed by the addition of 2 vol% glycerol, and stirring again for 4 h. Then, 10 mg of N-CDs particles were added, and the mixture was sonicated for 1 h. The resulting mixture was designated as the CS / N-CDs suspension. Finally, the mixture was poured into a petri dish (length × width × height: 12 cm × 12 cm × 2 cm) and placed in a 60 °C oven for 6 h to obtain the nanocomposite film.

[0081] The obtained CS / N-CDs thin films were subjected to performance tests, and the test results are as follows:

[0082] Figure 1 FTIR spectra and barrier and antioxidant properties of CS / N-CDs thin films. (A) FTIR spectrum of the thin film; (B) UV-Vis absorption spectrum of the thin film; (C) Water vapor permeability; (D) Oxygen permeability; (E) Carbon dioxide permeability; (F) DPPH- free radical scavenging activity. Figure 1 It can be seen that the functional groups of the chitosan-based film did not undergo characteristic changes after incorporating different proportions of N-CDs, indicating that N-CDs and CS have good compatibility. When exposed to ultraviolet radiation from 200 nm to 800 nm, the composite film's ability to absorb ultraviolet light increases with the increase of N-CDs content, thus enabling the film to effectively block ultraviolet light from entering food. The presence of N-CDs gives the CS / N-CDs film a good barrier effect against water vapor, carbon dioxide, and oxygen, and can also improve the film's antioxidant activity.

[0083] Figure 2 The antibacterial properties of CS / N-CDs films were investigated. (A) Effects of the films on the growth of *Escherichia coli* and *Staphylococcus aureus* under darkness and ultraviolet light; (B) Relative inhibition rate of the films against *Escherichia coli*; (C) Relative inhibition rate of the films against *Staphylococcus aureus*. Figure 2It can be seen that the antibacterial ability of the composite film continuously increases with the increase of N-CDs incorporation. This is because N-CDs are zero-dimensional NMs with high specific surface area, which increases the contact between the film and the bacterial cell wall, thereby causing mechanical damage to the bacteria. In addition, N-CDs contain amide and amino groups, making them positively charged, which can combine with the negative potential of the bacterial surface, leading to severe damage to the bacterial cell membrane, leakage of intracellular substances, and ultimately bacterial death.

[0084] Example 2

[0085] The application of CS / N-CDs suspension in improving the preservation ability of chili peppers includes the following steps:

[0086] The CS / N-CDs suspension immersion method involves wiping the fruit after soaking and then air-drying it, followed by storage at room temperature.

[0087] The CS / N-CDs suspension spraying method involves spraying the suspension onto the chili pepper fruit (calculated to yield 1.25 mL of spray solution per chili pepper), air-drying, and then storing at room temperature.

[0088] All treated fruits were harvested, photographed, and their weight loss rate was recorded on days 0, 7, 14, 21, and 28.

[0089] Comparative Example 1 (CK)

[0090] The CS / N-CDs suspension in Example 2 is omitted and replaced with pure water; otherwise, it remains the same as in Example 2.

[0091] Comparative Example 2CaCl2

[0092] The CS / N-CDs suspension in Example 2 was omitted and replaced with a 2% CaCl2 solution; otherwise, it remained the same as in Example 2.

[0093] Comparative Example 3Fe-P NMs

[0094] Weigh 0.5 g of polyvinylpyrrolidone (PVP), 2.025 g of ferric chloride (III) hexahydrate (FeCl3·6H2O), and 3.45 g of ammonium dihydrogen phosphate (NH4H2PO4) and dissolve them in 10 mL, 20 mL, and 20 mL of deionized water, respectively. Mix the obtained PVP solution and NH4H2PO4 solution in a 250 mL round-bottom flask. Place the FeCl3 solution in a separatory funnel and add it dropwise to the mixed solution at a rate of one drop every 6 seconds, while stirring with a magnetic stirrer throughout the process. After the addition is complete, continue stirring for 30 min. Transfer all the turbid liquid after stirring to a centrifuge tube and centrifuge at 6000 rpm for 10 min. Remove the supernatant and transfer the precipitate to a 100 mL stainless steel high-pressure reactor lined with polytetrafluoroethylene. Place the reactor at 180 °C and react for 6 h. After cooling, centrifuge at 5000 rpm for 5 min to collect the precipitate and wash it three times with deionized water. Finally, the powder was dried at 60°C for 2 hours in a vacuum drying oven to obtain iron-phosphorus nanomaterial powder.

[0095] Then, 10 mg / L of Fe-P NMs was applied to chili peppers according to the application method in Example 2.

[0096] Figure 3 The effects of immersion in a CS / N-CDs suspension on (A) phenotype and (B) weight loss of pepper fruits during storage. Figure 3 As can be seen from the physical appearance photographs, the peppers treated with CK, Fe-P NMs suspension, and CaCl2 suffered severe microbial damage, while the peppers treated with CS / N-CDs suspension maintained a good physical appearance throughout the 21 days of storage. To further evaluate the preservation effect of CS / N-CDs on peppers, the weight loss rate, a freshness indicator, was measured. All treatments showed increased weight loss during storage, which was related to moisture loss and nutrient consumption. After 21 days of storage, the peppers treated with CS / N-CDs immersion had the lowest weight loss rate at 8.5%, significantly lower than the 23.8% of CK.

[0097] Figure 4 This study depicts the changes in microorganisms in pepper fruits after immersion in a CS / N-CDs suspension during storage. (A) Relative abundance of dominant bacterial communities at the phylum level; (B) Relative abundance of the main genera of the dominant bacterial communities. Figure 4It can be seen that among all the pepper fruits treated with immersion, the dominant bacterial phyla were Proteobacteria (81.4%), Bacteroidetes (5.4%), Firmicutes (5.2%), and Actinobacteria (6.9%). Compared with the control group, the abundance of Actinobacteria in pepper fruits treated with CS / N-CDs suspension increased by 355.4%, while the abundance of Proteobacteria decreased by 5.1%. At the genus level, the abundance of Escherichia coli and Aureobacterium, which can cause pepper fruit rot, decreased by 98.2% and 65.0%, respectively, in the CS / N-CDs suspension treatment compared with the control group. In addition, compared with the CK group, the abundance of Pseudomonas, Arthrobacter and Sphingobacterium in pepper fruits treated with CS / N-CDs suspension increased by 61.4%, 96.9% and 92.8% respectively. These microorganisms can be used as biocontrol agents to effectively prevent fruit diseases.

[0098] Figure 5 The effects of spraying a CS / N-CDs suspension on (A) phenotype and (B) weight loss of pepper fruits during storage. Figure 5 It can be seen that during the 21 days of storage, no mold appearance or brown spots were observed in the CS / N-CDs suspension treatment, and the peppers maintained better texture compared to other treatments. In contrast, significant microbial damage and extensive mold growth were observed in peppers treated with CK, Fe-P NMs, and CaCl2. The weight loss of the sprayed peppers during storage was consistent with that of the immersion treatment. On the 21st day of storage, the weight loss rate of the CS / N-CDs suspension (7.3%) was significantly lower than that of the CK group (16.0%).

[0099] Figure 6 The changes in microorganisms during the storage of pepper fruits after spraying with CS / N-CDs suspension. (A) Relative abundance of dominant bacterial communities at the phylum level; (B) Relative abundance of the main genera of the dominant bacterial communities. From Figure 4It can be seen that among all the sprayed pepper fruits, the dominant bacterial phyla were Proteobacteria (74.9%), Bacteroidetes (3.4%), Firmicutes (9.2%), and Actinobacteria (9.6%). Compared with the control (CK), the abundance of Bacteroidetes in pepper fruits treated with CS / N-CDs suspension increased by 10.5%, while the abundance of Proteobacteria decreased by 12.1%. At the genus level, compared with the CK, the abundance of *Escherichia coli* and *Aureobacter*, which cause fruit rot, and *Rhodotorula*, which causes fruit shriveling, decreased by 82.9%, 91.8%, and 72.6%, respectively, in pepper fruits treated with CS / N-CDs suspension. Furthermore, compared to the control group, the CS / N-CDs suspension treatment increased the abundance of beneficial bacteria genera such as *Pseudomonas* and *Arthrobacter* (which can act as biocontrol agents) by 87.1% and 88.7%, respectively, while the abundance of *Sphingobacterium* and *Paenibacillus* (which can attack fungal cell walls) increased by 95.3% and 99.9%, respectively. In addition, the abundance of *Ochrobactrum* in pepper fruits treated with the CS / N-CDs suspension increased by 95.0% compared to the control group; *Ochrobactrum* can limit bacterial scab disease in pepper and tomato fruits.

[0100] In summary, CS / N-CDs suspension immersion or spraying treatments have a good preservation effect on pepper fruits, reducing weight loss during storage. The treatments downregulate microorganisms associated with fruit rot and shriveling (Enterobacter, Chryseobacterium, and Ralstonia) while upregulating beneficial microorganisms (Pseudomonas, Arthrobacter, Sphingobacterium, Paenibacillus, and Ochrobactrum). These microorganisms can act as biocontrol agents to effectively prevent fruit diseases and provide a favorable microecological environment for pepper fruit storage.

[0101] Comparative Example 4

[0102] Comparison of quality ratio optimization between CS and N-CDs

[0103] The 10 mg N-CDs particles in Example 1 were replaced with 0, 2, and 50 mg N-CDs particles, respectively, while other conditions remained the same as in Example 1, resulting in 0%, 0.1%, and 2.5% CS / N-CDs suspensions and films, respectively.

[0104] from Figure 2 It can be seen that the antibacterial ability of the composite film continuously increases with the increase of N-CDs incorporation. This is because N-CDs are zero-dimensional NMs with high specific surface area, which increases the contact between the film and the bacterial cell wall, thereby causing mechanical damage to the bacteria. In addition, N-CDs contain amide and amino groups, making them positively charged, which can combine with the negative potential of the bacterial surface, leading to severe damage to the bacterial cell membrane, leakage of intracellular substances, and ultimately bacterial death.

[0105] Example 3

[0106] Pepper fruits were packaged in 0.5% CS / N-CDs film and stored at room temperature. All treated fruits were removed, photographed, and weight loss was recorded on days 0, 7, 14, 21, and 28.

[0107] Comparative Example 5CK

[0108] The chili peppers are not treated in any way.

[0109] Comparative example 6CS membrane

[0110] The CS / N-CDs film in Example 3 is omitted and replaced with a CS film, while everything else remains the same as in Example 3.

[0111] Comparative example 7PE film

[0112] The CS / N-CDs film in Example 3 is omitted and replaced with a PE film (commonly used in the market), while everything else remains the same as in Example 3.

[0113] Figure 7 This is a performance comparison between Example 3 and Comparative Examples 5-7. From... Figure 7It can be seen that on the 21st day of storage, the peppers without film packaging (CK) and those treated with PE showed browning, rotting, and mold, while the peppers packaged with CS / N-CDs film showed lower maturity and no moldy appearance. The CK and PE groups began to rot on the 7th day of storage and were completely rotten by the 21st day. The peppers packaged with CS film also showed microbial damage after 21 days of storage. In contrast, the peppers packaged with CS / N-CDs film showed no obvious signs of rot. Next, the weight loss and firmness of peppers without film packaging and those packaged with different films were compared. On the 21st day of storage, the weight loss rate of peppers packaged with CS / N-CDs film was 67.2%, lower than the 84.5% of the CK group and the 77.2% of the CS group, respectively. The peppers packaged with PE film showed the lowest weight loss rate at 18.1%. PE film packaging showed the lowest weight loss among all treatments due to its ultra-low permeability. However, this characteristic of PE film makes the peppers susceptible to microbial infection and rot, which is fully verified in the photographs of their physical appearance. Firmness is another key parameter determining the deterioration of pepper fruit quality. Firmness decreases with prolonged storage time. On the 21st day after storage, the firmness of peppers without film packaging, those packaged in CS (Chemical Solids) film, and those packaged in CS / N-CDs film decreased by 4.8 times, 4.1 times, and 2.5 times, respectively, compared to the beginning of storage. On the 21st day after storage, the firmness of peppers packaged in CS / N-CDs film increased by 100% and 45.5% compared to the CK (Control Group) and CS group, respectively.

[0114] In summary, CS / N-CDs film has a good preservation effect on chili peppers. Its antibacterial properties can protect chili peppers from microbial damage. On the other hand, CS / N-CDs film has excellent barrier properties against ultraviolet rays, water, carbon dioxide and oxygen.

[0115] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preserving chili peppers by regulating the distribution of microorganisms, characterized in that, The method involves using a chitosan / nitrogen-doped carbon dot composite system to down-regulate the relative abundance of microorganisms related to the decay and shriveling of chili fruits and up-regulate the relative abundance of beneficial microorganisms. The method involves applying the chitosan / nitrogen-doped carbon dot composite system to chili fruits by impregnation or spraying. When used in maceration, the microorganisms associated with fruit rot and shriveling include: Enterobacteriaceae (… Enterobacter ) and Chlorella ( Chryseobacterium Beneficial microorganisms include: Pseudomonas ( ); Pseudomonas Arthrobacter spp. Arthrobacter ) and Sphingosine Bacillus ( Sphingobacterium ); When applying the spray, the microorganisms associated with fruit rot and shriveling include: Enterobacteriaceae (… Enterobacter ), Chlorella ( Chryseobacterium ) and Rolstonia ( Ralstonia ) ， Beneficial microorganisms include: Pseudomonas ( ) Pseudomonas Arthrobacter spp. Arthrobacter ), Sphingomyelin spp. Sphingobacterium ) 、 Bacillus spp. ( Paenibacillus ) and the genus *Pseudomonas* ( Ochrobactrum ); The preparation method of the chitosan / nitrogen-doped carbon dot composite system includes: A carbon source and a nitrogen source were dispersed in water and subjected to a hydrothermal reaction. After the reaction was completed, the mixture was cooled, filtered through a membrane, and freeze-dried to obtain nitrogen-doped carbon dots. Chitosan was dispersed in an acetic acid solution, followed by the addition of glycerol and mixing. The resulting nitrogen-doped carbon dots were then added and stirred to form a chitosan / nitrogen-doped carbon dot composite system. The carbon source was citric acid or its hydrate; the nitrogen source was urea; the mass ratio of the carbon source to the nitrogen source was 1:1.5; the hydrothermal reaction conditions were 150-200℃ for 3-8 hours; and the mass ratio of the nitrogen-doped carbon dots to chitosan was 0.005:

1.

2. The method of claim 1, wherein The soaking process involves immersing the fruit in a suspension of chitosan / nitrogen-doped carbon dots, wiping it after soaking, air-drying it, and storing it at room temperature. The spraying process involves spraying the chitosan / nitrogen-doped carbon dot suspension onto the chili pepper fruit, air-drying it, and storing it at room temperature.

3. The method of claim 1, wherein The carbon source was dispersed at a concentration of 0.1 g / mL in water.

4. The method of claim 1, wherein In the preparation method of the chitosan / nitrogen-doped carbon dot composite system, the dispersion concentration of chitosan in acetic acid solution is 0.02 g / mL.

5. A method for reducing weight loss of chili peppers during storage using the chitosan / nitrogen-doped carbon dot composite system as described in claim 1, characterized in that, The method involves applying a chitosan / nitrogen-doped carbon dot composite system to chili pepper fruits via impregnation or spraying.

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