Antibacterial carbon dots based on coordination functionalization and preparation method and application thereof

Abstract

This invention discloses a coordination-functionalized antibacterial carbon dot, its preparation method, and its application, belonging to the field of novel nanomaterial antibacterial technology. The antibacterial carbon dot (TM-CDs) is prepared from theanine and malic acid via microwave heating. The TM-CDs exhibit a chelation adsorption capacity of at least 150 mg / g for ferric ions. The carbon dot surface possesses numerous oxygen-containing groups that can bind with ferric ions to form stable complexes. Furthermore, its chelation capacity and rate for ferric ions are stronger than that of the heptaphilin secreted by *Pseudomonas berries*, competitively chelating ferric ions around *Pseudomonas berries* and inhibiting its growth. This invention also provides a method for applying this antibacterial carbon dot in the preservation of aquatic products, particularly salmon. The TM-CDs can inhibit the growth of various dominant spoilage bacteria in aquatic products, exhibiting good biocompatibility and superior antibacterial effect, showing promising application prospects in the food preservation field.

Description

Technical Field

[0001] This invention belongs to the field of novel antibacterial agent technology, specifically relating to a metal coordination functionalized antibacterial carbon dot, a method for preparing the antibacterial carbon dot, and the application of the carbon dot in the preservation of aquatic products, especially in the preservation of fresh salmon meat. Background Technology

[0002] Iron is an essential element for the growth and development of almost all bacteria. It participates in numerous bacterial biological processes, such as the tricarboxylic acid cycle, electron transport chain, oxidative phosphorylation, nitrogen fixation, and the biosynthesis of aromatic compounds. It also participates in the synthesis of metabolites such as porphyrins, toxins, antibiotics, cytochromes, pigments, and heptaphosphates. Peroxidases, catalases, and superoxide dismutases maintain cells by eliminating harmful free radicals; they also contain iron. Furthermore, iron cations act as cofactors for many enzymes, directly participating in and influencing cell composition. In iron deficiency, DNA and / or RNA synthesis decreases, bacterial growth and sporulation are inhibited, leading to morphological changes in bacteria.

[0003] Although the Earth's crust is rich in iron, its biological use in aerobic environments is impossible due to the low solubility of iron(III). Therefore, bacteria have evolved methods to remove and absorb iron from living organisms (plants and animals), freshwater, seawater, and soil. One of the main methods for bacterial survival and iron uptake under iron-limited conditions is the production of low-molecular-weight iron-chelating molecules, known as siderophores, for iron absorption. Siderophores produced by bacteria in iron-scarce conditions have a higher affinity for ferric ions (Fe3+) than for ferrous ions (Fe2+), thus facilitating iron uptake from iron-scarce environments. If bacterial siderophores cannot absorb ferric ions from their surrounding environment, most bacteria will perish.

[0004] Carbon dots (CDs), as a novel type of carbon-based nanomaterial, have attracted considerable attention from researchers in materials science, optics, biology, medicine, and food safety in recent years due to their diverse physicochemical properties, good biocompatibility, unique optical properties, extremely low cost, good eco-friendliness, abundant functional groups, high stability, and high electron mobility. In the field of food safety, particularly in antibacterial research, carbon dots are an important raw material for the preparation of carbon dot-based nanozymes. Carbon dots or carbon dot-based nanozymes can inhibit bacterial growth or even kill bacteria by promoting the production of reactive oxygen species, damaging bacterial cell walls and membranes, or inhibiting bacterial DNA and RNA transcription. This type of antibacterial method targets the bacteria themselves, and with prolonged use, bacteria easily develop drug resistance.

[0005] For example, prior art CN 114259005A discloses a method for preparing carbon dots with antibacterial properties. This method involves dissolving glucose, biogenic amine, and sodium chloride in a specific mass ratio, then treating the mixture with a hydrothermal reaction, followed by centrifugation, purification, and drying to obtain the final product. Another example is prior art CN 114304249A, which discloses an antibacterial carbon dot based on onions, its preparation method, and its application in the preservation of aquatic products. This antibacterial carbon dot is obtained by mixing onions and water in a specific ratio, followed by a hydrothermal reaction, alcohol precipitation, purification, and drying. Although the raw materials are inexpensive and readily available, and the preparation process is relatively simple, its minimum inhibitory concentration (MIC) against *Pseudomonas* is only 2 mg / mL.

[0006] Aquatic products are considered a high-quality source of nutrition due to their rich content of protein and fatty acids. However, this high nutrient content and high water content also make fresh aquatic products more prone to spoilage than other foods. Studies have shown that during the spoilage of aquatic products, one or more microorganisms initially exist in low numbers, but as the spoilage process progresses, their reproduction rate increases and their putrefactive ability strengthens, gradually dominating in the final stages of spoilage and becoming the dominant or specific spoilage organisms (SSOs). Among these, psychrophilic microorganisms such as *Pseudomonas* spp., *Acinetobacter* spp., and *Shewanella putrefaciens* are specific spoilage organisms for aquatic products under refrigerated conditions. *Pseudomonas berries* exhibits strong cold resistance and can maintain a high growth rate at low temperatures. This makes *Pseudomonas berryae* a dominant microbial community in cold-stored foods, primarily found in meat and meat products, milk and dairy products, and aquatic products under refrigeration, and capable of becoming a common specific spoilage bacterium in these foods. In chilled aquatic products, such as salmon, tuna, and rainbow trout, *Pseudomonas berryae* is the main specific spoilage bacterium. Currently, antibacterial methods against *Pseudomonas berryae* are not widespread, such as natural antibacterial agents, chemically synthesized antibacterial films, and modified atmosphere storage, and their antibacterial effects are not particularly prominent or significant. Summary of the Invention

[0007] (a) Technical problems to be solved

[0008] This invention aims to solve one of the following technical problems existing in the prior art or related technologies:

[0009] To address the problems of existing antibacterial agents primarily targeting the bacteria themselves, which can easily lead to bacterial resistance, and the insufficient antibacterial effect of antibacterial carbon dots, this invention employs a technical approach different from the existing antibacterial carbon dot principle to prepare a more effective antibacterial carbon dot and applies it to the preservation of aquatic products.

[0010] (II) Technical Solution

[0011] To address the aforementioned technical problems, this invention provides a method for preparing antibacterial carbon dots based on coordination functionalization and their application in the preservation of aquatic products. The specific technical solution adopted is as follows:

[0012] A type of antibacterial carbon dot TM-CDs based on coordination functionalization is characterized by being prepared by microwave heating using theanine and malic acid as raw materials. The antibacterial carbon dot TM-CDs inhibits the growth of various dominant aquatic spoilage bacteria.

[0013] Preferably, the dominant aquatic spoilage bacteria include *Pseudomonas fragi* (P. fragi), *Pseudomonas fluorescens* (P. fluorescens), *Aeromonassobria* (A. sobria), *Hafinia alvei* (H. alvei), and *Serratia marcescens* (S. marcescens).

[0014] More preferably, the minimum inhibitory concentrations against *Pseudomonas berryensis*, *Pseudomonas fluorescens*, *Aeromonas sobrio*, *Havnia vesicae*, *Aeromonas serrata*, and *Pseudomonas berryensis* are not higher than 0.25 mg / mL, 0.50 mg / mL, 0.25 mg / mL, 0.50 mg / mL, and 0.50 mg / mL, respectively.

[0015] Preferably, the antibacterial carbon dots TM-CDs have a chelation adsorption capacity for ferric ions of not less than 150 mg / g, thereby inhibiting bacterial growth and reproduction by competitively adsorbing ferric ions with the ferrophiles secreted by bacteria.

[0016] Preferably, the malic acid is at least one of D-malic acid, L-malic acid and DL-malic acid; and the theanine is L-theanine.

[0017] The preparation method of the above-mentioned antibacterial carbon dots TM-CDs provided by the present invention is as follows: First, prepare a theanine solution; then add malic acid to the prepared theanine solution to form a mixed solution; after mixing the mixed solution, microwave treatment is performed, and after microwave treatment, the solution is cooled to obtain a viscous liquid; after dissolving the obtained viscous liquid, it is separated and freeze-dried to obtain antibacterial carbon dots TM-CDs.

[0018] Preferably, the steps of the method are as follows:

[0019] 1) Dissolve theanine in deionized water to prepare a theanine solution with a concentration of 0.8g / 10mL-1.2g / 10mL;

[0020] 2) Dissolve malic acid in the theanine solution obtained in step (1) and mix the resulting mixture thoroughly; wherein the concentration of malic acid in the mixture is 0.8 g / 10 mL;

[0021] 3) Microwave the solution obtained in step (2), and cool it to room temperature after microwave treatment to obtain a viscous liquid;

[0022] 4) Add deionized water to the viscous liquid obtained in step (3), filter after fully dissolving, dialyze the filtrate, and freeze dry to obtain antibacterial carbon dots TM-CDs.

[0023] Preferably, the microwave treatment in step (3) has the following characteristics: microwave power: 300W-800W; treatment time: 2min-8min.

[0024] Preferably, step (4) uses a 0.22μm water filter for filtration; dialysis uses a dialysis bag of 200Da-500Da and is performed for 4 hours.

[0025] Preferably, the application of any of the above-mentioned antibacterial carbon dots TM-CDs in the preservation of aquatic products.

[0026] More preferably, the above application refers to its application in the preservation of chilled fish and meat.

[0027] More preferably, the fish meat is salmon, tuna, or rainbow trout.

[0028] Preferably, the above application method is as follows: under aseptic conditions, the fish meat is cut into pieces, the surface is dried under aseptic conditions, the fish pieces are immersed in the carbon dot™-CDs solution, soaked thoroughly, dried, and then stored in an aseptic preservation box. The concentration of the antibacterial carbon dot™-CDs solution is 0.50 mg / mL.

[0029] (III) Beneficial Effects

[0030] Compared with the prior art, the beneficial effects obtained by the present invention are as follows:

[0031] I. This invention employs a novel antibacterial principle that differs from existing methods targeting the bacterial cell itself, providing a new approach for the preparation of novel antibacterial agents. Based on the dependence of bacteria on ferrophosphate during contamination, a carbon dot with a stronger adsorption capacity for ferric ions is prepared, forming a competitive inhibition of ferric ions during bacterial growth, thereby inhibiting bacterial growth. The antibacterial principle employed is completely different from existing antibacterial principles targeting the bacterial cell and will not cause bacterial resistance. Specifically, this invention uses theanine and malic acid as raw materials to synthesize carbon dots TM-CDs through microwave heating. These carbon dots TM-CDs have excellent ferric ion chelating ability. The maximum ferric ion adsorption capacity is predicted to be 154.56 mg / g using the Langmuir isotherm model. Furthermore, the adsorption kinetic model demonstrates that the carbon dots have a stronger ferric ion chelating ability and rate than the ferrophosphate secreted by *Pseudomonas berries*, thus inhibiting its growth and reproduction. Meanwhile, actual measurements showed that the minimum inhibitory concentration (MIC) of the carbon dots TM-CDs against *Pseudomonas berryis* was 0.25 mg / mL, which is an order of magnitude higher than that of existing antibacterial carbon dots.

[0032] Second, after co-culturing mouse embryonic osteoblasts and rat blood cells with carbon dots TM-CDs, no inhibition of mouse embryonic osteoblast growth or destruction of rat erythrocytes was found, indicating that the carbon dots have good biocompatibility.

[0033] Third, by comparing the kinetic models of adsorption of ferric ions by carbon dots TM-CDs, carbon dots TM-CDs-ferrophile mixture and ferrophile, it can be demonstrated that carbon dots TM-CDs have a stronger chelating ability for iron than ferrophile and preferentially bind ferric ions.

[0034] IV. The carbon dot TM-CDs prepared in this invention possess excellent broad-spectrum antibacterial properties. When applied to the preservation of aquatic products, experimental results show that carbon dot TM-CDs can effectively extend the shelf life of salmon by 3-4 days, which is superior to the traditional food preservative—sodium dehydroacetate (1-2 days). Furthermore, these antibacterial carbon dot TM-CDs exhibit good biocompatibility and excellent antibacterial effect, showing promising application prospects in the field of aquatic product preservation. Attached Figure Description

[0035] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0036] Figure 1 This is a transmission electron microscope (TEM) image of the carbon dot TM-CDs prepared in Example 4.

[0037] Figure 2 The graph shows the cytotoxicity of the carbon dot TM-CDs prepared in Example 4 as a function of concentration.

[0038] Figure 3 The carbon dots TM-CDs prepared in Example 4 are effective against Fe 3+ Specific binding diagram.

[0039] In the figure, a shows the fluorescence measurement results after the mixture of 7 different metal ions and carbon dots TM-CDs; b shows the fluorescence measurement results of Fe. 3+ Fluorescence measurement results after mixing with six different metal ions in pairs and then reacting with carbon dots TM-CDs.

[0040] Figure 4 The carbon dot TM-CDs prepared in Example 4 are combined with Fe. 3+ The following is a comparison of infrared spectra.

[0041] Figure 5 The carbon dots TM-CDs prepared in Example 4 are combined with Fe 3+ The adsorption isotherm model.

[0042] Figure 6 The image shows a plate of CASAD assay for the ferrophile isolated and purified in Example 9. In the image, a is the plate of CASAD assay for ferrophile in the fermentation broth; b is the plate of CASAD assay for ferrophile after passing through XAD-2 macroporous resin; and c is the plate of CASAD assay for ferrophile after passing through XAD-2 macroporous resin and Sephadex LH-20.

[0043] Figure 7 The diagram shows the minimum inhibitory concentration of carbon dot TM-CDs prepared in Example 4 against various dominant aquatic spoilage bacteria.

[0044] Figure 8 The growth curves of *Pseudomonas berries* under different growth conditions are shown.

[0045] Figure 9 The carbon dots TM-CDs prepared in Example 4 and the ferrophile isolated and purified in Example 9 regarding the chelation of Fe 3+ Adsorption kinetics model diagram of the adsorption capacity.

[0046] Figure 10 The effect of carbon dot TM-CDs prepared in Example 4 and the traditional food preservative sodium dehydroacetate on the shelf life (total bacterial count) of salmon. Detailed Implementation

[0047] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0048] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0049] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0050] Various improvements and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, which will be obvious to those skilled in the art.

[0051] Unless otherwise specified, the materials, reagents, instruments, etc. used in the following examples are all conventional materials, reagents, and instruments in the art, and can be obtained by those skilled in the art through commercial channels.

[0052] Example 1

[0053] This embodiment uses malic acid as a base material, reacting it with theanine, chlorogenic acid, lactic acid, arginine, oxalic acid, caffeic acid, ascorbic acid, lysine, spermidine, and ethylenediaminetetraacetic acid, respectively. The specific experimental procedures are as follows:

[0054] (1) Weigh 1.2g of the above raw materials (i.e., chlorogenic acid, lactic acid, etc.) and dissolve them in 10mL of deionized water.

[0055] (2) Add 0.8g of L-malic acid to the solution in step (1) and sonicate at room temperature for 8 minutes to avoid clumping.

[0056] (3) Transfer the saturated solution obtained in step (2) to a 50 mL conical flask and seal it. Heat at 800 W for 2 min. After the reaction is complete, cool to room temperature to obtain a viscous liquid.

[0057] (4) Add 20 mL of deionized water to the viscous liquid in step (3), dissolve it completely, filter the solution with a 0.22 μm aqueous filter, dialyze the filtered solution with a 500 Da dialysis bag for 5 h, and then freeze dry to obtain the desired carbon dot sample.

[0058] To verify the antibacterial properties and Fe chelation of the 10 samples obtained by the above method 3+ The ability to inhibit the growth of *Pseudomonas berryae* and chelate Fe in 10 carbon point samples. 3+ The capabilities were measured (see Examples 10 and 8 below for specific measurement methods), and the results are as follows:

[0059] Table 1. Inhibition of *Pseudomonas fructans* growth by carbon dots synthesized from malic acid and several other substances, and the chelation of Fe... 3+ Results of ability

[0060]

[0061] Note: "+" indicates the ability to inhibit the growth of *Pseudomonas berrieseri*; "-" indicates the ability not to inhibit the growth of *Pseudomonas berrieseri*; "√" indicates the ability to chelate Fe. 3+ Ability; "×" indicates lack of ability to chelate Fe 3+ ability.

[0062] Experimental results showed that malic acid and ethylenediaminetetraacetic acid could not synthesize carbon dots, while the others could synthesize carbon dots with fluorescent properties. However, as can be seen from Table 1, the carbon dots synthesized by the above substances and malic acid did not have the function of chelating ferric ions, and did not have or only had a weak ability to inhibit the growth of Pseudomonas berries.

[0063] Example 2

[0064] This embodiment provides a method for preparing chelated ferric ion carbon dots TM-CDs, the specific steps of which are as follows:

[0065] (1) Weigh 0.8g of L-theanine and dissolve it in 10mL of deionized water.

[0066] (2) Add 0.8g of DL-malic acid to the solution in step (1) and sonicate at room temperature for 5 minutes to avoid clumping.

[0067] (3) Transfer the saturated solution obtained in step (2) to a 50 mL conical flask and seal it. Heat at 400 W for 5 min. After the reaction is complete, cool to room temperature to obtain a viscous liquid.

[0068] (4) Add 10 mL of deionized water to the viscous liquid in step (3), dissolve it completely, filter the solution with a 0.22 μm water filter, dialyze the filtered solution with a 200 Da dialysis bag for 4 h, and freeze dry to obtain chelated ferric ion carbon dots TM-CDs.

[0069] Example 3

[0070] This embodiment provides a method for preparing chelated ferric ion carbon dots TM-CDs, the specific steps of which are as follows:

[0071] (1) Weigh 1.0g of L-theanine and dissolve it in 10mL of deionized water.

[0072] (2) Add 0.8g of D-malic acid to the solution in step (1) and sonicate at room temperature for 10 minutes to avoid clumping.

[0073] (3) Transfer the saturated solution obtained in step (2) to a 50 mL conical flask and seal it. Heat at 300 W for 8 min. After the reaction is complete, cool to room temperature to obtain a viscous liquid.

[0074] (4) Add 15 mL of deionized water to the viscous liquid in step (3), dissolve it completely, filter the solution with a 0.22 μm water filter, and then dialyze the filtered solution with a 500 Da dialysis bag for 3 h before freeze drying to obtain chelated ferric ion carbon dots TM-CDs.

[0075] Example 4

[0076] This embodiment provides a method for preparing chelated ferric ion carbon dots TM-CDs, the specific steps of which are as follows:

[0077] (1) Weigh 1.2g of L-theanine and dissolve it in 10mL of deionized water.

[0078] (2) Add 0.8g of L-malic acid to the solution in step (1) and sonicate at room temperature for 8 minutes to avoid clumping.

[0079] (3) Transfer the saturated solution obtained in step (2) to a 50 mL conical flask and seal it. Heat at 800 W for 2 min. After the reaction is complete, cool to room temperature to obtain a viscous liquid.

[0080] (4) Add 20 mL of deionized water to the viscous liquid in step (3), dissolve it completely, filter the solution with a 0.22 μm water filter, dialyze the filtered solution with a 500 Da dialysis bag for 5 h, and then freeze dry to obtain chelated ferric ion carbon dots TM-CDs.

[0081] Example 5

[0082] This example demonstrates the cytotoxicity of the carbon dot TM-CDs prepared in Example 4. The effect of carbon dot TM-CDs on the proliferation activity of MC3T3-E1 cells was detected using the CCK-8 assay. MC3T3-E1 cells were cultured at 5 × 10⁶ cells per well. 3 Cells were seeded into 96-well plates. The cell culture plates were incubated overnight at 37°C in a 5% CO2 incubator. Different concentrations of carbon dots were prepared using complete culture medium (90% α-MEM medium + 10% fetal bovine serum) (100 μL added to each well, concentrations of 1.00, 0.75, 0.50, 0.25, and 0.00 mg / mL, three parallel experiments). The mixture was thoroughly mixed, and the cells were incubated at 37°C in a 5% CO2 incubator for 24 h. The supernatant was discarded, and 10 μL of CCK-8 working solution was added to each well for staining. The cells were then incubated for another 1 h. The OD value of each well was read using a microplate reader at λ = 450 nm, and the cell proliferation rate was calculated. The experimental results showed that at the MIC concentration (i.e., 0.25 mg / mL), the cell proliferation rate of this carbon dot could reach 97.14% (e.g., 90% α-MEM medium + 10% fetal bovine serum). Figure 2 The result indicates that the carbon dot is not harmful to MC3T3-E1 cells and is safe.

[0083] Example 6

[0084] This embodiment measures the specificity of the carbon dot TM-CDs prepared in Example 4 for the selection of ferric ions. The measurement method is as follows:

[0085] Configure 6mM Na + K + Mg 2+ Ca 2+ Cu 2+ Al 3+ and Fe 3+ The above metal solution was mixed with an equal volume of 0.5 mg / mL carbon dots TM-CDs and reacted on a shaker at 100 r / min at room temperature for 6 h; the above Fe removal solution was then... 3+ Other metal ions and Fe 3+ The mixtures were combined in pairs and then mixed with an equal volume of 0.5 mg / mL carbon dots TM-CDs, under the same reaction conditions as described above. The results are as follows. Figure 3 As shown in the figure, fluorescence measurements indicate that carbon dots TM-CDs have a specific binding ability for ferric ions.

[0086] Example 7

[0087] This embodiment investigates the changes in the infrared spectrum of the carbon dot TM-CDs prepared in Example 4 after chelating ferric ions.

[0088] A 6 mM solution of ferric ions was mixed with 0.5 mg / mL carbon dots TM-CDs, and the reaction conditions were the same as in Example 5. Carbon dots TM-CDs and carbon dots TM-CDs-Fe 3+ The mixture was freeze-dried into a powder, and infrared spectroscopy analysis revealed a 1544 cm⁻¹ value. -1 The C=O coordinate at that location has been redshifted to 1595 cm. -1 Location; 1375cm -1 The OH red shifted to 1408 cm⁻¹ -1 Location. 917 cm of carbon dots TM-CDs. -1 OH is treated with the addition of Fe 3+ The sensual cluster then disappeared. (At 595cm) -1 A new functional group, Fe-O, is formed at this site. The above experimental results indicate that the oxygen-containing groups in carbon dots TM-CDs can combine with ferric ions, leading to changes in the original functional groups of the carbon dots TM-CDs. The oxygen-containing groups in TM-CDs combine with Fe... 3+ Coordinate bonds are formed. Specifically, the O atoms on the TM-CDs surface bond to the Fe... 3 + The donation of lone pairs of electrons leads to Fe 3+ Coordinate bonds are formed between the O atom and the O atom.

[0089] Example 8

[0090] This embodiment measures the ability of carbon dot TM-CDs prepared in Example 4 to chelate ferric ions. The measurement method is as follows:

[0091] 0.1 mg / mL of carbon dots TM-CDs were reacted with Fe at initial concentrations of 5, 10, 15, 30, 50, 70, and 100 ppm. 3+ The solutions were mixed, and the reaction conditions were the same as in Example 6. The Fe content at each initial concentration was determined using a Shimadzu ICPE-9820 inductively coupled plasma atomic emission spectrometer. 3+ The equilibrium concentration, according to the formula q e =(C0-C e The equilibrium adsorption capacity is calculated by multiplying V / M. To better understand the relationship between carbon dots TM-CDs and Fe... 3+ The mechanism of action was investigated, and the Langmiur and Fruedlich isotherm models for this adsorption experiment were evaluated. Experimental results show that the Langmiur isotherm model provides a better fit than the Fruedlich isotherm model (e.g., ...). Figure 5 (and Table 2), illustrating Fe 3+ The adsorption sites on the carbon dot surface are a monolayer and uniform, indicating that the adsorption sites on the carbon dot will not adsorb more Fe after saturation. 3+The parameters of the two models were obtained through linear fitting, and the Fe content of carbon dots TM-CDs was... 3+ The maximum adsorption capacity was 154.56 mg / g. The R values ​​for the Langmiur isotherm model and the Fruedlich isotherm model were... 2 The values ​​are 0.9942 and 0.9692, respectively.

[0092] Table 2 shows the fitting results of the Langmiur isotherm model and the Fruedlich isotherm model.

[0093]

[0094] Example 9

[0095] This embodiment describes the isolation and purification of the ferrophile secreted by *Pseudomonas berries*, as preparation for the experiment in Example 12. The experimental method is as follows:

[0096] The liquid culture medium for *Pseudomonas berries* was MKB liquid medium, and the culture conditions were 28℃ and 160 r / min. The fermentation broth was centrifuged at 10000 rpm for 15 min and filtered through a 0.22 μm filter to obtain a sterile supernatant. The fermentation filtrate was mixed with XAD-2 macroporous resin at a ratio of 2:1 and adsorbed overnight at 20℃ and 120 r / min. The adsorbed XAD-2 macroporous resin was separated from the fermentation filtrate, and an appropriate volume of 75% methanol / water was added. The mixture was then packed into a column and eluted with 75% methanol / water at a flow rate of 1.5 mL / min (twice the column volume). After elution, the collected sample was evaporated to dryness in a rotary evaporator at 50℃ and 80 r / min, and then dissolved in sterile deionized water. The sample solution was added to Sephadex LH-20 for further separation and purification. The eluent was 10% methanol / water, and the flow rate was 1.5 mL / min. After elution, the collected sample was evaporated to dryness in a rotary evaporator at 50 °C and 80 r / min. Then, sterile deionized water was added to dissolve the sample, and the solution was freeze-dried to obtain a viscous solid, which was the purified ferrophosphate. Ferrophilic solutions from fermentation broth, ferrophosphate after passing through XAD-2 macroporous resin, and ferrophosphate solutions after passing through XAD-2 macroporous resin and Sephadex LH-20 were respectively added to CASAD detection plates. Ferrophilic rings were obtained for the three solutions, indicating that the proportion of ferrophosphate was significantly increased after separation and purification (e.g., ...). Figure 6 ).

[0097] Example 10

[0098] In this embodiment, the minimum inhibitory concentration of the carbon dot TM-CDs prepared in Example 4 against *Pseudomonas berryae*, *Pseudomonas fluorescens*, *Pseudomonas tempera*, *Havnia vesicae*, and *Aeromonas serrata* was determined by plate counting method.

[0099] The aforementioned dominant aquatic spoilage bacteria were cultured in a shaker at 28℃ until the logarithmic growth phase. The bacteria were washed and diluted, and 200 μL of the bacterial suspension was inoculated into fresh LB broth. Different concentrations (0, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL) of carbon dots TM-CDs were added to the broth. After culturing for 12 h, the culture was plated onto LB nutrient agar plates using the plate method and incubated at 28℃ for 12 h. Colony growth was observed, and the MIC (micron limit) of each dominant aquatic spoilage bacteria was obtained. Figure 7 ).from Figure 7 It can be seen that the minimum inhibitory concentrations of the carbon dots TM-CDs prepared in Example 4 against *Pseudomonas berryensis*, *Pseudomonas fluorescens*, *Pseudomonas tempera*, *Havnia vesicae*, and *Aeromonas serrata* are 0.25 mg / mL, 0.50 mg / mL, 0.25 mg / mL, 0.50 mg / mL, and 0.50 mg / mL, respectively.

[0100] Example 11

[0101] This embodiment investigates the effect of the growth and reproduction of *Pseudomonas berries* on Fe... 3+ The dependence of the carbon dots TM-CDs prepared in Example 4 and the evidence that they chelate the Fe surrounding the bacteria 3+ It has an inhibitory effect on its growth. The specific experimental steps are as follows:

[0102] *Pseudomonas berryae* was cultured in a shaker at 28°C until it reached the logarithmic growth phase. The bacteria were then washed and diluted, and four tubes of fresh LB broth were prepared. *Pseudomonas berryae*, *Pseudomonas berryae* + Fe were added sequentially to each tube. 3+ *Pseudomonas berryans* + TM-CDs and *Pseudomonas berryans* + Fe 3+ +TM-CDs. Added Fe 3+ The volume was 100 μL, the concentration was 2 μM (non-toxic to *Pseudomonas berryans*); the volume of TM-CDs added was 1 mL, the concentration was 0.25 mg / mL. The growth curves of *Pseudomonas berryans* under four different growth conditions are shown below. Figure 8 As shown. From Figure 8 It is known that the growth of *Pseudomonas berries* requires Fe. 3+ Carbon dots TM-CDs can block the iron absorption of this bacterium by chelating ferric ions, thereby causing *Pseudomonas berrieseri* to die due to lack of nutrients.

[0103] Example 12

[0104] This example compares the ability of carbon dots TM-CDs prepared in Example 4 to chelate ferric ions with the ferrophile isolated and purified in Example 9.

[0105] Carbon dots TM-CDs, ferrophile, and a mixture of carbon dots TM-CDs and ferrophile were added to a ferric ion solution, respectively, under the same reaction conditions as in Example 6. The concentrations of carbon dots were 0.1 mg / mL, ferrophile was 0.143 mg / mL, and ferric ions were 5 ppm. The equilibrium adsorption capacity q of ferric ions at different time points was determined using an ICPE-9820. t The adsorption kinetics model was used to fit the chelation process of the three substances with ferric ions, and the results showed that all three followed a pseudo-first-order model. Figure 8 As shown in Table 3, carbon dot TM-CDs bind to ferric ions faster and in greater quantities than heparin. This indicates that when *Pseudomonas berryensis* and carbon dot TM-CDs coexist, TM-CDs interfere with the iron absorption of the bacterium, preventing *Pseudomonas berryensis* from growing normally.

[0106] Table 3. Kinetic model fitting results for carbon dot TM-CDs, ferrophiles, and mixtures of carbon dot TM-CDs and ferrophiles.

[0107]

[0108] Example 13

[0109] This embodiment uses the carbon dot TM-CDs prepared in Example 4 to determine their effect on the preservation of salmon flesh. Specifically, the total bacterial count of salmon flesh at different shelf lifespans was measured. The specific experimental and measurement procedures are as follows:

[0110] Fresh salmon fillets purchased from the local market were transported to the laboratory under refrigeration. The fillets were aseptically cut into small pieces, aseptically dried, and then soaked in different treatment solutions for 10 minutes. After draining until dry, they were stored in sterilized fresh containers. The salmon fillets were divided into three groups: (1) control group; (2) sodium dehydroacetate group; and (3) TM-CDs group. The concentration of sodium dehydroacetate was 0.5 mg / mL, and the concentration of TM-CDs was 0.5 mg / mL. All samples were stored at 4°C. The storage period was 12 days, and the total number of colonies (TVC) of the samples was measured daily for each storage day. Specifically, the number of colonies in the salmon samples was determined using the plate pouring method. 10 g of salmon back fillet was accurately weighed, and 90 mL of 0.90% sterile physiological saline was added and gently tapped for 3 minutes. 100 μL of the sample was evenly spread on a plate and serially diluted 10-fold. Incubate at 28°C for 24 hours, record the number of colonies, and calculate the TVC value.

[0111] Seafood, being rich in nutrients, easily becomes a nutrient source for microorganisms. Total bacterial count is an important indicator for evaluating the degree of microbial contamination in food. Generally, a TVC value greater than 6 log CFU / g indicates that salmon has begun to rot and is no longer edible. Figure 10 As shown, the TVC value of fresh salmon was 1.64 log CFU / g, and the TVC value increased with prolonged storage time. The graph shows that the TVC value of salmon in the TM-CDs group was consistently lower than that of the control group and the sodium dehydroacetate group, indicating that TM-CDs have good antibacterial properties. On day 6, the TVC value of the control group was 6.14 ± 0.095 log CFU / g, indicating that the control group had begun to rot, and the salmon in the sodium dehydroacetate group was about to enter the rot stage. However, the salmon in the TM-CDs group was in good condition and showed no signs of rot. On day 9, the TVC values ​​of the control group and the sodium dehydroacetate group showed that the salmon had rotted, while the TVC value of the salmon in the TM-CDs group was 5.96 ± 0.166 log CFU / g, indicating that the salmon was about to enter the rot stage. The curve in the graph shows that the salmon in the TM-CDs group entered the rot stage on day 10. Therefore, TM-CDs can extend the shelf life of salmon by 3-4 days, while sodium dehydroacetate can extend it by 1-2 days. This indicates that the antibacterial carbon dots TM-CDs effectively inhibit bacterial damage to fish, and their antibacterial performance is far superior to that of the traditional food preservative sodium dehydroacetate. Therefore, these antibacterial carbon dots TM-CDs have excellent application prospects in the field of food preservation.

[0112] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A type of antibacterial carbon dots TM-CDs based on coordination functionalization, characterized in that, It is prepared by microwave heating using theanine and malic acid as raw materials. The antibacterial carbon dots TM-CDs inhibit the growth of various dominant aquatic spoilage bacteria. The preparation method of the antibacterial carbon dots TM-CDs is as follows: (1) Dissolve theanine in deionized water to prepare a theanine solution with a concentration of 0.8 g / 10mL - 1.2 g / 10mL; (2) Dissolve malic acid in the theanine solution obtained in step (1) and mix the resulting mixture evenly; wherein the concentration of malic acid in the mixture is 0.8 g / 10 mL; (3) The solution obtained in step (2) is subjected to microwave treatment, and then cooled to room temperature to obtain a viscous liquid; the microwave treatment: microwave power: 300 W - 800 W; treatment time: 2 min ~ 8 min; (4) Add deionized water to the viscous liquid obtained in step (3), filter after fully dissolving, dialyze the filtrate, freeze dry to obtain antibacterial carbon dots TM-CDs.

2. The antibacterial carbon dots TM-CDs based on coordination functionalization according to claim 1, characterized in that, The dominant spoilage bacteria in aquatic products include *Pseudomonas fructosa* (… Pseudomonas fragi, P. fragi ), fluorescent pseudomonas ( Pseudomonas fluorescens, P. fluorescens Aeromonas hydrophila ( ), Aeromonas hydrophila Aeromonas sobria, A.sobria ), Hafnia apiae ( Hafinia alvei , H. alvei Aeromonas serrata ( ), Aeromonas sarrella ( Serratia marcescens, S. marcescens ).

3. The antibacterial carbon dots TM-CDs based on coordination functionalization according to claim 2, characterized in that, The minimum inhibitory concentrations against *Pseudomonas berryae*, *Pseudomonas fluorescens*, *Aeromonas sobrio*, *Havnia vesicae*, and *Aeromonas serrata* were not higher than 0.25 mg / mL, 0.50 mg / mL, 0.25 mg / mL, 0.50 mg / mL, and 0.50 mg / mL, respectively.

4. The antibacterial carbon dots TM-CDs based on coordination functionalization according to claim 1, characterized in that, It has a chelation and adsorption capacity of no less than 150 mg / g for ferric ions, and inhibits bacterial growth and reproduction by competitively adsorbing ferric ions with the ferrophile secreted by bacteria.

5. The antibacterial carbon dots TM-CDs based on coordination functionalization according to claim 1, characterized in that, In step (4) of the preparation method, filtration is carried out using a 0.22μm water filter; dialysis is carried out using a 200Da-500Da dialysis bag and dialysis treatment is performed for 4 hours.

6. The application of the antibacterial carbon dots TM-CDs described in any one of claims 1-4 in the preservation of aquatic products.

7. The application according to claim 6, characterized in that, Application in the preservation of chilled fish and meat.

Images