Preparation method and application of eel carbon dots with antioxidant activity

Abstract

The application belongs to the technical field of biomass nanomaterial preparation and application, and discloses a preparation method of eel carbon dots with antioxidant activity and application thereof. In view of the problems of poor stability of existing natural antioxidants, low added value of traditional eel processing waste and easy pollution of the environment, the application proposes to use natural eel waste as the only raw material, and to use high-temperature and high-pressure hydrothermal reaction at 180 DEG C to 220 DEG C to promote the complete depolymerization and carbonization of dense collagen and bone matrix. The process realizes in-situ doping of N element, overcomes the defect of easy toasting of animal source material, and the obtained eel carbon dots have excellent water solubility and low cytotoxicity, and exhibit extremely strong and stable antioxidant activity. The application not only realizes the high-value utilization of marine biological waste, but also provides a green and economic solution for developing a new, safe and efficient nanoscale antioxidant.

Description

Technical Field

[0001] This invention belongs to the field of food preservation technology, specifically relating to a method for preparing and applying eel carbon dots with antioxidant activity. Background Technology

[0002] Reactive oxygen species (ROS, such as hydroxyl radicals and superoxide anions) are unavoidable byproducts of metabolism in organisms. Appropriate amounts of ROS play a role in cell signaling and immune responses, but excessive ROS can lead to oxidative stress, resulting in DNA damage, lipid peroxidation, and protein denaturation. This is closely related to aging and various diseases, such as cancer, cardiovascular disease, and neurodegenerative diseases. Furthermore, in the food industry, oxidation is one of the main causes of food spoilage, flavor loss, and reduced nutritional value. Therefore, developing efficient and safe antioxidants is of great significance for food preservation and the medical and health fields.

[0003] Currently, antioxidants on the market are mainly divided into two categories: synthetic antioxidants such as BHA and BHT, and natural antioxidants such as vitamin C, vitamin E, and polyphenols. While synthetic antioxidants are low-cost and have significant antioxidant effects, they carry potential toxicity and carcinogenic risks. Long-term intake may damage organs such as the liver, and their application in the food and pharmaceutical fields is subject to increasingly stringent regulations. Traditional natural small-molecule antioxidants are highly safe, but often suffer from poor stability, poor water solubility, low bioavailability, and increased costs.

[0004] Carbon dots (CDs), as a novel zero-dimensional carbon nanomaterial, have attracted widespread attention due to their small size (typically less than 10 nm), excellent water solubility, chemical stability, biocompatibility, low toxicity, and tunable surface functional groups. Studies have found that carbon dots synthesized with specific precursors possess significant electron or hydrogen donor capabilities, effectively scavenging free radicals and exhibiting excellent antioxidant activity, making them a promising next-generation nano-antioxidant. However, current carbon dot preparation relies on chemical reagents such as citric acid and ethylenediamine, resulting in high costs and potential chemical residues. The preparation conditions for some carbon dots are demanding and cumbersome, and the quantum yield or antioxidant activity of the products is often low, requiring the introduction of additional surface modifiers or heteroatom doping (such as nitrogen and sulfur doping) to improve performance. Eel is a nutrient-rich biomass resource, rich in high-quality protein, peptides, polysaccharides, and various trace elements. Its processing often generates large amounts of byproducts or underutilized low-value components, leading to resource waste and environmental pressure.

[0005] In summary, developing a carbon dot that uses eel waste as raw material, has a simple and environmentally friendly preparation process, and possesses excellent water solubility, high stability, and strong antioxidant activity, has significant technical value and application prospects for replacing traditional synthetic antioxidants and solving the problem of poor stability of natural antioxidants. Summary of the Invention

[0006] Based on this, the present invention provides a method for preparing eel carbon dots with antioxidant activity and its application. The method mainly uses eel processing waste as a carbon source to prepare carbon dots through a one-step hydrothermal method. The preparation process is simple, which realizes the reuse of aquatic product processing waste and has potential application prospects in the field of food preservation.

[0007] The objective of this invention is to provide a method for preparing eel carbon dots with antioxidant activity, comprising the following steps: S1. Hydrothermal reaction: Eel powder is dissolved in an aqueous solution at a mass ratio of 1:5, and a hydrothermal reaction is carried out. The hydrothermal reaction time is 6h-10h, and the temperature is 180℃-220℃, to obtain a brown solution. S2. Concentration and extraction for defatting: The brown solution obtained in step S1 is concentrated, and the concentrate is extracted with a weakly polar or moderately polar organic solvent for defatting. After extraction, the organic phase is removed, and the aqueous phase solution is collected to obtain a defatted solution. S3. Multi-stage purification and freeze-drying: The defatted solution obtained in step S2 is centrifuged, filtered through a filter membrane, dialyzed, and freeze-dried to obtain eel carbon dot freeze-dried powder.

[0008] Preferably, the eel powder in step S1 is made by drying and grinding eel waste, wherein the eel waste is one or more of eel skin, eel head, eel tail or eel viscera, the drying temperature is 55℃-65℃, and the grinding time is 10min-15min.

[0009] Preferably, the organic solvent in step S2 is one or more of ethyl acetate, dichloromethane, petroleum ether, n-hexane, diethyl ether, or methyl tert-butyl ether.

[0010] Preferably, the concentration method in step S2 is one or more of low-temperature vacuum concentration, nanofiltration membrane separation concentration, or rotary evaporation concentration.

[0011] Preferably, the concentration method in step S2 is rotary evaporation concentration, the temperature of the rotary evaporation is 45℃-55℃, and the endpoint of the rotary evaporation is 1 / 8 to 1 / 12 of the volume of the remaining original solution.

[0012] Preferably, the centrifugation rate in step S3 is 8000-10000 rpm, the centrifugation time is 15-30 min; the pore size of the filter membrane is 0.22 μm, the pore size of the dialysis bag is 1000 Da, the dialysis time is 48 h, and the freeze-drying time is 48 h.

[0013] The eel carbon dots with antioxidant activity prepared by any of the above preparation methods.

[0014] The second objective of this invention is to provide the application of the above-mentioned eel carbon dots in food preservation. Preferably, the food is fish balls, and the prepared fish balls are soaked in an aqueous solution of eel carbon dots, wherein the concentration of the aqueous solution is 0.4 mg / mL.

[0015] The technical principles of each step in this invention are as follows: Eel powder is rich in biomass organic components such as protein, amino acids, unsaturated fatty acids, and polypeptides. Under high-temperature hydrothermal and high-pressure water conditions (180℃-220℃), it undergoes a series of reactions including dehydration, carbonization, polymerization, and aromatization. On one hand, the biomass precursor gradually pyrolyzes and rearranges to form a carbon dot framework with a nano-carbon core structure. The well-organized carbon core is the carrier for the carbon dots to stably exert their antioxidant effects. On the other hand, during the reaction, a large number of polar active functional groups such as hydroxyl (-OH), carboxyl (-COOH), and amino (-NH2) groups are enriched on the surface of the carbon dots. These functional groups are the core active sites for the antioxidant activity of carbon dots, which can provide hydrogen protons, chelate metal ions, and scavenge DPPH· and ABTS in vivo. + The invention utilizes reactive free radicals such as hydroxyl groups to achieve antioxidant effects. The hydrothermal reaction temperature for preparing eel carbon dots must be maintained within the range of 180℃-220℃, and the reaction time must be maintained between 6h and 10h. If the reaction temperature is too low or the time is too short, the eel biomass carbonization will be insufficient, failing to form a complete carbon dot structure, resulting in a low amount of active functional groups and weak antioxidant activity. If the temperature is too high or the time is too long, it will lead to over-carbonization of the carbon dots, structural agglomeration, and a large amount of removal and degradation of surface active functional groups, resulting in the loss of active sites and the formation of inert carbides with no antioxidant activity, significantly reducing the overall antioxidant performance. The optimal feed-to-liquid ratio of 1:5 ensures that the eel biomass is fully dispersed and reacts uniformly, avoiding uneven local carbonization and ensuring a uniform loading of active functional groups.

[0016] Step S2, extraction and defatting, is crucial for removing interfering impurities, purifying active components, and locking in antioxidant activity. It directly avoids the inhibitory effect of impurities on the antioxidant performance of carbon dots and is a necessary pretreatment step to ensure high activity of the finished product. The brown crude solution after the hydrothermal reaction in Step S1 contains, in addition to the target antioxidant carbon dots, a large amount of eel endogenous oil, fat-soluble impurities, incompletely reacted fat-soluble organic matter, and carbonization byproducts. These fat-soluble impurities have no antioxidant activity and can also coat the surface of carbon dots, obscuring active functional groups, hindering contact between carbon dots and free radicals, and are prone to oxidative deterioration, indirectly reducing the overall antioxidant effect. Ethyl acetate, a fat-soluble organic solvent, can specifically extract oil and fat-soluble impurities from the solution and completely remove them by removing the organic phase. A gentle rotary evaporation temperature of 45℃-55℃ avoids high-temperature damage to the heat-sensitive antioxidant functional groups on the carbon dot surface. If the rotary evaporation temperature is too high (e.g., >60℃), these surface-active groups are prone to secondary dehydration condensation during prolonged heating. If the temperature is below 45℃, reasonable evaporation efficiency cannot be guaranteed. Simultaneously, at 45-55℃ and a vacuum of -0.08 to -0.1 MPa, gentle and stable evaporation can be achieved, preventing boiling over caused by complex matrices. The final solution ratio at the rotary evaporation endpoint is a key indicator for controlling the stability of carbon dot colloids and preventing aggregation. A suitable final solution ratio allows for the appropriate enrichment of the active components of the carbon dots while avoiding excessive concentration that could lead to carbon dot aggregation and deactivation, thus maximizing the preservation and optimization of the antioxidant activity of the carbon dots.

[0017] Step S3 removes ineffective impurities, purifies the carbon dot structure, and solidifies antioxidant activity, directly determining the purity and activity stability of the finished carbon dots. The defatted solution still contains uncarbonized small-molecule inorganic salts, water-soluble proteins, small-molecule byproducts, and trace large-particle aggregates. These impurities lack antioxidant activity and interfere with the free radical scavenging ability of the carbon dots, reducing the purity and activity of the finished product. In the multi-stage purification process, centrifugation removes large solid residues and agglomerated carbon particles; membrane filtration retains regularly sized active carbon dots and removes minute impurities; dialysis precisely removes small-molecule water-soluble impurities and ions, maximizing carbon dot purity; and finally, freeze-drying uses a low-temperature vacuum dehydration method, unlike high-temperature drying, which completely preserves the active functional groups such as hydroxyl and carboxyl groups on the carbon dot surface, avoiding functional group degradation and carbon dot structure collapse caused by high temperatures. Simultaneously, it solidifies the nanostructure of the carbon dots, ensuring that the finished eel carbon dots possess stable and long-lasting antioxidant activity.

[0018] Compared with existing technologies, the present invention, by adopting the above-mentioned technical solution, has the following effective effects: This invention uses waste generated during eel processing as a carbon source to prepare carbon dots. By utilizing industrial waste instead of expensive chemical reagents, it significantly reduces raw material costs and solves the environmental pollution problem caused by eel processing waste, aligning with the principles of green chemistry and a circular economy. The protein-rich eel waste provides an ample natural nitrogen source. During carbonization, nitrogen is in-situ incorporated into the carbon framework, significantly increasing the electron cloud density of the carbon dots without the need for additional toxic nitrogen sources such as ethylenediamine, thus endowing them with stronger free radical scavenging capabilities than ordinary biomass carbon dots. The prepared eel waste carbon dots not only retain the biocompatibility of natural components but also overcome the shortcomings of traditional natural antioxidants, such as poor heat resistance and easy decomposition, exhibiting excellent water solubility and light / thermal stability. Attached Figure Description

[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Figure 1 The image is a TEM image of the eel-based carbon dots prepared in Comparative Example 1 of this invention at a hydrothermal temperature of 160°C and a hydrothermal time of 4 hours.

[0021] Figure 2 The image shown is a TEM image of the eel-based carbon dots prepared in Comparative Example 2 of this invention at a hydrothermal temperature of 240°C and a hydrothermal time of 12 hours.

[0022] Figure 3 This represents the free radical scavenging rate of eel carbon dots in Example 1 of the present invention. Wherein, (a) represents DPPH·, and (b) represents ABTS. + ·, (c) is OH·.

[0023] Figure 4 This represents the free radical scavenging rate of eel carbon dots in Example 2 of the present invention. Wherein, (a) represents DPPH·, and (b) represents ABTS. + ·, (c) is OH·.

[0024] Figure 5 This represents the free radical scavenging rate of eel carbon dots in Example 3 of the present invention. Wherein, (a) represents DPPH·, and (b) represents ABTS. + ·, (c) is OH·.

[0025] Figure 6 The images show the particle size distribution and TEM image of carbon dots in eel from Example 2 of this invention. (a) is the particle size distribution image, and (b) is the TEM image.

[0026] Figure 7 This is the XPS energy spectrum of carbon dots in eel from Example 2 of the present invention; Figure 8 The infrared spectrum of carbon dots in eel according to Embodiment 2 of the present invention; Figure 9 This is a graph showing the changes in eel carbon-dot treated fish balls during storage at 4°C in Example 2 of the present invention. In the graph, (a) represents pH, (b) represents water holding capacity, (c) represents malondialdehyde content, and (d) represents volatile basic nitrogen content. Detailed Implementation

[0027] 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.

[0028] 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.

[0029] 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.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0034] Example 1 The method for preparing eel carbon dots with antioxidant activity described in this embodiment includes the following steps: S1. Crush eel processing waste such as eel skin, eel head, eel tail or eel viscera, dry and grind into powder at 55℃ for 15 minutes; S2. Using the eel powder obtained in step S1 as raw material, dissolve it in an aqueous solution at a mass ratio of 1:5. After hydrothermal reaction, the hydrothermal reaction time is 6 hours and the temperature is 180℃, a brown solution is obtained. S3. The brown solution obtained in step S2 is subjected to rotary evaporation at a temperature of 45°C. The rotary evaporation endpoint is set at 1 / 8 of the original solution volume. The solution is then extracted with ethyl acetate 10 times to remove the organic phase. The aqueous phase solution is collected to obtain the defatted solution. The carbon dot precursor of this invention is a hydrothermal product with an extremely high water content (approximately 90% or more). The evaporation rate of water under vacuum depressurization conditions is positively correlated with temperature. When the temperature is below 45°C, the latent heat of vaporization of water is insufficient, resulting in an extremely slow evaporation rate. This not only leads to a significant increase in production cycle and energy consumption, which is not economically viable for industrial production, but more importantly, the carbon dots remain in a heated state in the liquid phase for extended periods. Even at lower temperatures, this can induce slow hydrolysis of surface ligands, affecting batch stability. The reason why nano-carbon dots can exist stably in water is due to the repulsive force of like charges from their surface charge and the barrier effect of the surface hydration film. When the volume of the concentrated solution is too small and the concentration is too high, the distance between nanoparticles shortens drastically, causing the hydration film to rupture. This leads to irreversible aggregation and precipitation of the carbon dots. Once aggregation occurs, the active sites are deeply buried within the framework, resulting in a precipitous drop in antioxidant activity, and the carbon dots cannot be redispersed in water, thus losing their value as a preservative coating.

[0035] S4. The defatted solution obtained in step S3 is centrifuged at a speed of 8000 rpm for 20 min, filtered through a 0.22 μm filter membrane, dialyzed through a 1000 Da dialysis bag for 48 h, and then freeze-dried for 48 h to obtain freeze-dried eel carbon dot powder with antioxidant activity.

[0036] Example 2 S1. Crush eel processing waste such as eel skin, eel head, eel tail or eel viscera, dry and grind it into powder at 65℃ for 10 minutes; S2. Using the eel powder obtained in step S1 as raw material, dissolve it in an aqueous solution at a mass ratio of 1:5. After hydrothermal reaction, the hydrothermal reaction time is 8 hours and the temperature is 200℃, a brown solution is obtained. S3. The brown solution obtained in step S2 is subjected to rotary evaporation at a temperature of 50°C. The rotary evaporation endpoint is set at 1 / 10 of the original solution volume remaining. The solution is then extracted with ethyl acetate 10 times to remove the organic phase. After extraction, the aqueous phase solution is collected to obtain the defatted solution. S4. The defatted solution obtained in step S3 is centrifuged at a speed of 9000 rpm for 20 min, filtered through a 0.22 μm filter membrane, dialyzed through a 1000 Da dialysis bag for 48 h, and then freeze-dried for 48 h to obtain freeze-dried eel carbon dot powder with antioxidant activity.

[0037] Example 3 S1. Crush eel processing waste such as eel skin, eel head, eel tail or eel viscera, dry and grind into powder at 60℃ for 15 minutes; S2. Using the eel powder obtained in step S1 as raw material, dissolve it in an aqueous solution at a mass ratio of 1:5. After hydrothermal reaction, the hydrothermal reaction time is 10 hours and the temperature is 220℃, a brown solution is obtained. S3. The brown solution obtained in step S2 is subjected to rotary evaporation at a temperature of 55°C. The rotary evaporation endpoint is defined as 1 / 12 of the original solution volume remaining. The solution is then extracted with ethyl acetate 10 times to remove the organic phase. The aqueous phase solution is collected to obtain the defatted solution. Since the precursor of this invention is derived from eel processing waste (fish skin, fish bones, etc.), the solution contains trace amounts of incompletely carbonized polypeptides, protein hydrolysates, and lipid residues rich in double bonds. When the rotary evaporation temperature exceeds 55°C, under localized overheating and reduced pressure, it easily induces thermal denaturation of the protein and generates a large amount of foam, leading to severe bumping, direct product loss, and making the experiment impossible to proceed normally. The purpose of the concentration step is to reduce the solution volume to allow for efficient subsequent purification (such as dialysis or ultrafiltration). If the concentration ratio exceeds the upper limit (i.e., the solution is still very dilute), the dialysis efficiency will be extremely low and the dialysis time will be indefinitely prolonged due to the small concentration difference between the internal and external phases during subsequent dialysis purification. This would result in a huge liquid volume for subsequent freeze-drying or application, consuming enormous amounts of electrical energy and time, which is completely impractical for industrial applications.

[0038] S4. The defatted solution obtained in step S3 is centrifuged at a speed of 10,000 rpm for 20 min, filtered through a 0.22 μm filter membrane, dialyzed through a 1000 Da dialysis bag for 48 h, and then freeze-dried for 48 h to obtain freeze-dried eel carbon dot powder with antioxidant activity.

[0039] Comparative Example 1 When the hydrothermal temperature for preparing eel-based carbon dots is 160℃ and the hydrothermal time is 4h, the TEM image of the resulting product is as follows. Figure 1 As shown. By Figure 1As can be seen, the product mainly presents as a large area of ​​light gray amorphous polymer network matrix, doped with scattered polymer wrinkles, and no independent and clearly morphologically distinct nano-carbon dots were observed. This indicates that under these mild conditions, the dehydration and carbonization of the precursor was severely insufficient, and the reaction system only reached the cross-linking polymerization stage of the precursor, failing to achieve the aggregation and growth of carbon nuclei. This is because the product lacks sp... 2 The conjugated carbon core active center constructed by hybridization has extremely weak electron transfer or resonance stabilization ability for free radicals, resulting in a very low free radical scavenging rate and failing to exhibit the expected biological activity.

[0040] Comparative Example 2 When the hydrothermal temperature for preparing eel-based carbon dots was increased to 240℃ and the hydrothermal time was extended to 12h, the TEM image of the resulting product was as follows. Figure 2 As shown in the figure, the product mainly consists of large and irregularly shaped carbon nanoparticles. This is because the excessively high reaction temperature and long reaction time provide excess reaction energy, leading to excessive condensation and dehydration carbonization of the eel precursor. During this process, the oxygen-containing passivation groups on the carbon core surface undergo thermal degradation or loss, resulting in weakened electrostatic repulsion and steric hindrance effects between nanoparticles, severely disrupting colloidal stability and triggering non-specific aggregation and abnormal grain growth. This indicates that excessively high heat input will destroy the size confinement effect and uniform dispersion of carbon dots, making it impossible to obtain high-quality carbon nanoparticles with monodisperse characteristics.

[0041] Example 1: Determination of the in vitro antioxidant activity of carbon dots in eels (1) DPPH· free radical scavenging experiment Mix 100 μL of 0.1 mM DPPH ethanol solution with 100 μL of eel carbon dot solutions of different concentrations, and react at room temperature in the dark for 30 min. Measure the absorbance at 517 nm using a microplate reader. Calculate the DPPH free radical scavenging rate of CDs according to formula (1).

[0042] DPPH free radical scavenging rate (%) = (1) In the formula, Ai: absorbance of eel carbon dot solution + DPPH solution; Aj: absorbance of eel carbon dot solution + anhydrous ethanol solution; A0: absorbance of DPPH solution + anhydrous ethanol solution.

[0043] (2) ABTS + Free radical scavenging experiment ABTS stock solution (7 mmol / L) and potassium persulfate solution (2.45 mmol / L) were mixed 1:1 to prepare ABTS working solution. After standing in the dark for 14 h, the absorbance of the ABTS working solution at 734 nm was adjusted to 0.70 ± 0.02 with deionized water. 180 μL of ABTS working solution was mixed with 20 μL of CDs solution of different concentrations, and the mixture was stood at room temperature in the dark for 6 min. The absorbance was measured at 734 nm. The effect of CDs on ABTS was calculated according to formula (2). + • Free radical scavenging rate.

[0044] ABTS + • Free radical scavenging rate (%) = (2) In the formula, Ai: absorbance of CDs solution + ABTS working solution; Aj: absorbance of CDs solution + deionized water; A0: absorbance of ABTS working solution + deionized water.

[0045] (3) OH· free radical scavenging experiment 250 μL of eel carbon dot solutions of different concentrations were mixed with 250 μL of ferrous sulfate solution (9 mmol / L) and 250 μL of salicylic acid ethanol solution (9 mmol / L). Then, 250 μL of hydrogen peroxide solution (8.8 mmol / L) was quickly added and mixed thoroughly. The mixture was reacted at 37 °C for 30 min, cooled to room temperature, and the absorbance was measured at 510 nm. The CDs OH· free radical scavenging rate was calculated according to formula (3).

[0046] OH· free radical scavenging rate (%) = (3) In the formula, Ai: absorbance of eel carbon dot solution + reaction system solution; Aj: absorbance of eel carbon dot solution + reaction system solution (deionized water instead of hydrogen peroxide solution); A0: absorbance of deionized water + reaction system solution.

[0047] like Figure 3-5 As shown, with the gradual increase of carbon point concentration in eels, the effects of DPPH· and ABTS on DPPH· and ABTS· are observed. + The scavenging rates of · and OH· free radicals gradually increased in a dose-dependent manner. In particular, the mass concentrations of eel carbon dots calcined at 200°C for 8 hours that achieved half-maximal scavenging rates for all three free radicals were lower than the other two groups. For DPPH· and ABTS… + IC of · and OH· free radicals 50 The values ​​were 100.78, 132.73 and 2760.28 μg / mL, respectively.

[0048] Characterization of carbon dots in eel prepared in Example 2 of Example 2 The morphological characteristics, particle size, surface functional groups, and elemental composition of the carbon dots in the eel from Example 2 were analyzed, and the specific results are as follows: Figure 6-8 As shown.

[0049] Depend on Figure 6 It can be seen that the carbon dots in the eel are all approximately spherical in shape, with no aggregation observed, exhibiting obvious uniformity, indicating that they are soluble in water and dispersed evenly. Statistical analysis of the carbon dot particle size using ImageJ software shows that the particle size ranges from 2.41 to 5.93 nm, with an average particle size of 4.00 ± 0.70 nm. The overall particle size distribution is uniform, consistent with the morphological characteristics of carbon dots.

[0050] Figure 7 XPS spectra of eel carbon dots show that the main elemental composition of eel carbon dots is C, O and N, with relative contents of 67.79%, 19.67% and 12.54%, respectively. Figure 7 (b)-(d) High-resolution XPS images showing carbon dots C in eels 1s Mainly composed of CO, C=O, and CC groups; O 1s It mainly contains CO and C=O groups; N 1s It mainly consists of CN and NH groups.

[0051] Figure 8 The infrared spectrum of eel carbon spots shows that the eel carbon spots are located at 3415 cm⁻¹. -1 The strong absorption peak that appears is mainly caused by the stretching vibration of the OH group, at 2962 cm⁻¹. -1 The absorption peak that appeared was identified as the stretching vibration of the CH group, at 1659 cm⁻¹. -1 The absorption peak at 1396.5 cm⁻¹ can be attributed to the stretching vibration of the C=O group. -1 The absorption peak at 1455.1 cm⁻¹ may be related to the stretching vibration of the CN group. -1 The absorption peaks are mainly determined by the bending vibrations of the CH groups.

[0052] Detection Example 3: Application of eel carbon dots prepared in Example 2 in fish ball storage The prepared fish balls were soaked in an eel carbon point aqueous solution with a concentration of 0.4 mg / mL prepared in Example 2 for 2 hours at a temperature of 4°C. The pH, water holding capacity, malondialdehyde (MDA) content, and volatile basic nitrogen (TVB-N) content were measured. Figure 9 As shown in (a), during storage, the pH value of the fish balls in the eel carbon point group showed an upward trend compared to the control group. This was due to the decomposition of proteins by microorganisms and enzymes, producing alkaline nitrogenous substances. Figure 9As shown in (b), the overall water holding capacity of the eel carbon dot group of fish balls was slightly lower than that of the other two groups, but it decreased steadily and slowly throughout the storage period, with two plateau periods before and after. This group also exhibited the smallest decrease, indicating that the eel carbon dots greatly protected the integrity of the fish ball's protein gel structure, and the water loss was normal and slow, rather than a sudden loss caused by quality deterioration. Fat oxidation is the main cause of unpleasant flavor and quality decline in fish balls. Figure 9 As shown in (c), the MDA content in the eel carbon point group was consistently lower than that in the control group throughout the entire storage period. Furthermore, MDA was virtually undetectable on days 0 and 2, and even on day 8, the MDA content was only 0.53 mg / kg. This indicates that eel carbon points can effectively inhibit the initiation of the initial short lipid oxidation chain reaction and delay lipid hydrolysis and oxidation. Figure 9 As shown in (d), the TVB-N content in the eel carbon dot group increased from 0.93 mg / 100g to 2.8 mg / 100g on day 4 and then remained stable, indicating that the eel carbon dots played a certain inhibitory role in protein degradation, reducing protein oxidative damage by scavenging free radicals and maintaining the integrity of protein structure.

[0053] In summary, the carbon dots from eels provided by this invention have good antioxidant activity, which can delay lipid oxidation and protein oxidation in the storage and preservation of fish balls. They can be used as green coating nanomaterials in the preservation of aquatic products and have broad application potential in the field of aquatic product preservation. This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A method for preparing eel carbon dots with antioxidant activity, characterized in that, Includes the following steps: S1. Hydrothermal reaction: Eel powder is dissolved in an aqueous solution at a mass ratio of 1:5, and a hydrothermal reaction is carried out. The hydrothermal reaction time is 6h-10h, and the temperature is 180℃-220℃, to obtain a brown solution. S2. Concentration and extraction for defatting: The brown solution obtained in step S1 is concentrated, and the concentrate is extracted with a weakly polar or moderately polar organic solvent for defatting. After extraction, the organic phase is removed, and the aqueous phase solution is collected to obtain a defatted solution. S3. Multi-stage purification and freeze-drying: The defatted solution obtained in step S2 is centrifuged, filtered through a filter membrane, dialyzed, and freeze-dried to obtain eel carbon dot freeze-dried powder.

2. The preparation method according to claim 1, characterized in that, The eel powder mentioned in step S1 is made by drying and grinding eel waste, which is one or more of eel skin, eel head, eel tail or eel viscera. The drying temperature is 55℃-65℃ and the grinding time is 10min-15min.

3. The preparation method according to claim 1, characterized in that: The organic solvent in step S2 is ethyl acetate. dichloromethane petroleum ether n-Hexane Diethyl ether Or one or more of methyl tert-butyl ethers.

4. The preparation method according to claim 1, characterized in that: The concentration method described in step S2 is one or more of the following: low-temperature vacuum concentration, nanofiltration membrane separation concentration, or rotary evaporation concentration.

5. The preparation method according to claim 4, characterized in that: The concentration method described in step S2 is rotary evaporation concentration, wherein the rotary evaporation temperature is 45℃-55℃, and the rotary evaporation endpoint is 1 / 8 to 1 / 12 of the remaining original solution volume.

6. The preparation method according to claim 1, characterized in that: In step S3, the centrifugation rate is 8000-10000 rpm, the centrifugation time is 15-30 min, the pore size of the filter membrane is 0.22 μm, the pore size of the dialysis bag is 1000 Da, the dialysis time is 48 h, and the freeze-drying time is 48 h.

7. Eel carbon dots with antioxidant activity prepared by the preparation method according to any one of claims 1-6.

8. The application of the eel carbon dots according to claim 7, characterized in that, The eel carbon dots are used for food preservation.

9. The application according to claim 8, characterized in that, The food product is fish balls, which are prepared by soaking the fish balls in an aqueous solution of eel carbon dots, wherein the concentration of the aqueous solution is 0.4 mg / mL.

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