A method for preparing blue fluorescent antibacterial carbon dots based on glycosylation reaction

Abstract

This invention relates to a glycosylated blue fluorescent antibacterial carbon dot, its preparation method, and its applications. The glycosylated blue fluorescent antibacterial carbon dot is a nanoscale carbon material synthesized through a specific preparation method. Green, safe, and scalable glucose / fructose and bovine serum albumin are used as substrates for the Maillard reaction, providing the carbon source for the preparation of the glycosylated carbon dot. Fluorescent carbon dots with blue fluorescence and antibacterial activity are prepared in a bottom-up one-step hydrothermal reaction. Compared to existing methods, this preparation method is easy to operate, and the resulting product exhibits high brightness, stable fluorescence, and better biocompatibility, providing new possibilities for applications in biomedicine, food safety, and environmental protection. Furthermore, the preparation method is simple and easy to implement, and the raw materials can be obtained from natural resources, thus having a wide range of applications and broad technological prospects.

Description

Technical Field

[0001] This invention belongs to the field of food-derived glycosylated carbon dot preparation, specifically involving the preparation of blue fluorescent carbon dots with antibacterial properties using food-derived glycosylation reactions. Background Technology

[0002] Carbon dots (CDs) are an emerging type of carbon-based nanomaterial with unique physicochemical properties that demonstrate their potential applications in biomedicine, energy, and other fields. For example, carbon dots can be used as biolabels for bioimaging in fluorescence microscopy, exhibiting high brightness and stability; they can also be used in the fabrication of fluorescence sensors, demonstrating high selectivity and sensitivity for the detection of specific molecules. However, traditional carbon dot preparation methods require expensive precursors, strong acids, high temperatures, and long processing times, limiting their practical feasibility and scalability. In recent years, glycosylation has received widespread attention as a promising green synthetic strategy. Food-derived glycosylation is a common chemical reaction in food processing and cooking. It is caused by the reaction between sugar molecules in food and the amino groups of amino acids or proteins. Glycosylation can lead to changes in the color and flavor of food, and may also affect its nutritional value. In cooking and baking, glycosylation is often an important process for creating caramel color, oven flavor, and other characteristics in food. Furthermore, glycosylation can introduce other functional compounds during the reaction process, further expanding the performance of carbon dots. Glycosylated carbon dots are carbon dots prepared from carbohydrate compounds. The products have physiological activity, pigment coloring, and antibacterial properties.

[0003] However, glycosylated carbon dots have problems such as complex preparation, unclear fluorescence properties, and unclear antibacterial effects. Therefore, there is an urgent need to provide a method for preparing blue fluorescent antibacterial carbon dots based on glycosylation reaction that is convenient, quick, readily available, has stable fluorescence properties, and good antibacterial and bacteriostatic properties. Summary of the Invention

[0004] To address the aforementioned technical problems in the background art, this patent provides a method for preparing blue fluorescent antibacterial carbon dots based on glycosylation reactions. This method features green synthesis, simplicity, high efficiency, controllability, and strong scalability. This method can prepare carbon dots with excellent fluorescence and antibacterial properties at a relatively low cost and with minimal resource consumption. The prepared carbon dots can be applied in fields such as disinfection, medical device coatings, and food preservation, which is of great significance for improving people's quality of life and health.

[0005] The preparation method proposed in this invention first selects an easily obtainable and low-cost carbohydrate compound, such as glucose or fructose, as a precursor. Then, under certain temperature conditions, the carbohydrate is reacted with a specific carbon source (bovine serum albumin, BSA) to produce an intermediate product through glycosylation. Next, the intermediate product undergoes specific processing steps, such as filtration, dialysis, and drying, to obtain blue fluorescent antibacterial carbon dots.

[0006] The technical solution of this invention is as follows:

[0007] The first objective of this invention is to provide a method for preparing blue fluorescent antibacterial carbon dots based on glycosylation reaction, the method comprising the following steps:

[0008] (A) Mix carbohydrate compounds with bovine serum albumin (BSA), place in a hydrothermal reactor, and hydrothermally react at 120℃~180℃ for 6-12 hours;

[0009] (B) Dissolve and centrifuge the intermediate homogenate prepared in step (A), take the supernatant, dialyze, and freeze dry to obtain a yellowish-brown or dark yellow powder with a pungent odor, which is glycosylated carbon dot powder.

[0010] Furthermore, in step (A), the mass ratio of carbohydrates to bovine serum albumin is 1:1.

[0011] Furthermore, in step (A), the hydrothermal reaction involves mixing carbohydrate compounds with water at a mass-to-volume ratio of 1:10–40 (g / mL) and then reacting. Under these conditions, the blue fluorescence obtained from the glycosylation hydrothermal reaction is stable.

[0012] Furthermore, the carbohydrate compound mentioned in step (A) is one or more of glucose, fructose, sucrose, lactose, maltose, fructoside, glucosinolate, galactose, mannose, xylose, rhamnose, starch, cellulose, chitosan, and mannan.

[0013] Furthermore, step (A) of the hydrothermal reaction also includes controlling the pH value between 4 and 7.

[0014] Furthermore, the intermediate described in step (B) is a dark brown crude extract with a strong coffee roasted aroma.

[0015] Furthermore, the centrifugation conditions described in step (B) are 5000g for 20 minutes.

[0016] The second objective of this invention is to provide blue fluorescent antibacterial carbon dots based on glycosylation reaction prepared by the aforementioned preparation method.

[0017] A third objective of this invention is to provide the application of the aforementioned blue fluorescent antibacterial carbon dots based on glycosylation reactions in the preparation of antibacterial additives.

[0018] Furthermore, the application of the blue fluorescent antibacterial carbon dots based on glycosylation reaction in the preparation of antibacterial additives for meat products.

[0019] In a specific embodiment, the blue fluorescent antibacterial carbon dots are prepared into an antibacterial preservative solution for use in braised meat products. The technical solution of this invention has the following characteristics:

[0020] (1) Raw material selection: The selection of carbohydrate compounds as raw materials, such as glucose and fructose. These raw materials are usually characterized by abundant sources, low cost, and safety. In addition to glucose and fructose, other carbohydrate compounds can also be considered as raw materials. For example, sucrose, lactose, maltose, fructosides, and glucosides can also be used as raw materials for hydrothermal reactions. In addition, complex carbohydrates or polysaccharides can also be used as raw materials, such as starch, cellulose, chitosan, and mannan. These complex polysaccharides may be hydrolyzed into various monosaccharides during hydrothermal reactions, thereby generating different products. When selecting raw materials, attention should be paid to selecting raw materials with high purity (purity greater than 98%), accurately weighing 0.5 to 1 g, placing them in a 10 to 20 mL hydrothermal reactor, and dissolving them completely.

[0021] (2) Reaction Condition Control: The glycosylation reaction of this invention is carried out under high temperature conditions, specifically 120–180°C. At this temperature, the glycosylation reaction can simultaneously ensure reactivity and product stability, which is beneficial for carbon dot formation and can effectively limit undesirable side reactions. Appropriate acid-base conditions are crucial for the glycosylation reaction. Choosing a weakly acidic condition with a pH value between 4 and 7 is beneficial for the glycosylation reaction, which helps to improve the fluorescence and antibacterial properties of the product. In addition, weakly acidic conditions can also avoid excessive damage to the raw materials and help maintain the integrity of the raw materials. The reaction time of the glycosylation reaction is controlled within the range of 6–12 hours. Within this time range, it can fully ensure the reaction between the sugar compound and the carbon source, forming intermediate products and ultimately synthesizing blue fluorescent antibacterial carbon dots. Appropriate reaction time can ensure the stability and consistency of the product, while avoiding over-reaction that leads to product instability.

[0022] (3) Carbon Source Selection and Processing: BSA protein can serve as a natural carbon source, possessing abundant nitrogen functional groups and a robust protein structure, playing a crucial role in carbon dot preparation. Compared to other carbon sources, BSA protein exhibits excellent solubility and stability, is easy to handle, has no toxic side effects, and can be biodegraded, aligning with environmental protection principles. The selection and processing of the carbon source directly affect the performance of the final carbon dot product. For carbon sources like BSA protein, appropriate processing can enhance their reactivity and product stability during carbon dot preparation. Through heat treatment or chemical modification, the structure and functional groups of the carbon source can be controlled, making it more suitable for preparing carbon dots with specific properties, such as fluorescence and antibacterial properties.

[0023] (4) Reaction steps and mechanism:

[0024] 4.1 Selecting raw materials for glycosylation reaction: Select carbohydrate compounds (such as glucose, fructose, etc.) as raw materials for glycosylation reaction; select carbon source (such as BSA protein) as carbon source, and mix it with carbohydrate compounds in an appropriate solvent.

[0025] 4.2 Control reaction conditions: Place the mixed solution at a mild temperature, between 120 and 180 degrees Celsius, control the pH value under weakly acidic conditions (pH 4-7), and control the reaction time (6-12 hours).

[0026] 4.3 Glycosylation Mechanism: Glycosylation is a condensation reaction between carbohydrates and amino compounds (such as amino acids in proteins) to form N-glycosylated products. During this process, the aldehyde group (from the carbohydrate compound) condenses with the amino group (from the amino compound) to form a stable N-glycosylated structure. This invention uses food-grade carbohydrates as a carbon source to directly prepare glycosylated carbon dots using the glycosylation reaction. Glycosylation grafting is achieved during the glycosylation process, eliminating the need for pre-preparing carbon dots followed by the glycosylation reaction, making the preparation convenient, rapid, and readily available.

[0027] 4.4 Separation of intermediate products: The intermediate products are separated from the mixed solution after the reaction by appropriate separation steps (such as centrifugation (5000g, 20min), filtration (0.25um filter membrane) etc.).

[0028] 4.5 Dialysis and purification: The intermediate product is dialyzed to remove unreacted raw materials and byproducts, as well as impurities such as solvents, to obtain a pure intermediate product.

[0029] 4.6 Drying: The purified intermediate product was freeze-dried to obtain the final blue fluorescent antibacterial carbon dots.

[0030] (5) Characterization and analysis methods in a specific embodiment:

[0031] 5.1 Fluorescence spectrometer, used to detect the fluorescence properties of carbon dots. A fluorescence spectrometer can measure the fluorescence spectral characteristics of carbon dots, including excitation and emission spectra, thereby obtaining information such as the emission wavelength, fluorescence intensity, and fluorescence stability of the carbon dots.

[0032] 5.2 Ultraviolet-visible absorption spectrometer is used to measure the absorption spectral characteristics of carbon dots. It can obtain their absorption characteristics in the ultraviolet-visible region. Combined with fluorescence spectroscopy, it can provide a comprehensive understanding of the spectral performance of carbon dots.

[0033] 5.3 Antibacterial test: The prepared blue fluorescent antibacterial carbon dots were brought into contact with bacteria in the antibacterial test to observe their antibacterial effect and antibacterial mechanism, thereby evaluating the antibacterial performance of the carbon dots.

[0034] (6) Controlled Synthesis and Process Optimization: Adjusting the ratio of raw materials is one of the important means to achieve controlled synthesis. The ratio of carbon source to sugar compounds has a significant impact on the properties of the product. Appropriately adjusting the ratio of raw materials can control the size of carbon dots, fluorescence intensity, and antibacterial properties. The mass ratio of sugar to BSA is 1:1.

[0035] The technical solution of this invention has the following advantages over the prior art:

[0036] By developing applications of blue fluorescent antibacterial carbon dots in the aforementioned fields, the market demand for food microbial control and product preservation can be met, bringing broad business opportunities and economic benefits to enterprises.

[0037] 1. Food preservation

[0038] Application scenarios: Blue fluorescent antibacterial carbon dots have antibacterial properties and can be used as an additive in food packaging materials to extend the shelf life of food and enable food traceability management through fluorescent tracking technology.

[0039] Market demand: With the increasing demand for food safety and preservation, the demand for food preservation materials with antibacterial properties continues to rise.

[0040] Business Opportunity: The blue fluorescent antibacterial carbon dot material of this invention can be used as an antibacterial additive in food packaging to meet the market demand for food preservation and traceability management. Attached Figure Description

[0041] Figure 1 Flowchart for the preparation of glycosylated blue fluorescent antibacterial carbon dots;

[0042] Figure 2 Appearance of glycosylated fluorescent carbon dots under different preparation conditions: (A) hydrothermal conditions of 180℃ for 6 h; (B) hydrothermal conditions of 180℃ for 12 h.

[0043] Figure 3Ultraviolet-visible absorption spectroscopy characterization, (A) hydrothermal conditions: 180℃, 6h; (B) hydrothermal conditions: 180℃, 12h;

[0044] Figure 4 The glycosylated fluorescent carbon dots were characterized by fluorescence spectra under hydrothermal conditions of 180℃ for 6 h and 180℃ for 12 h.

[0045] Figure 5 CIE colorimetric images of glycosylated fluorescent carbon dots: (A) Glycosylated carbon dots at different concentrations (5.3400-0.0534 mg / mL) at 180℃ for 6 h; (B) Glycosylated carbon dots at different concentrations (0.9500-0.0095 mg / mL) at 180℃ for 12 h.

[0046] Figure 6 Antibacterial characterization of glycosylated fluorescent carbon dots. Detailed Implementation

[0047] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0048] Example 1: Preparation of glucose-derived glycosylated antibacterial carbon dots

[0049] In a high-pressure, polytetrafluoroethylene-lined hydrothermal reactor, 1 gram of glucose and 1 gram of BSA were mixed and dissolved in 10 mL of deionized water. The mixture was then sealed and placed in a muffle furnace and reacted at 180°C for 6 hours, followed by natural cooling to room temperature. This process yielded a brownish-black product. The reaction solution was then centrifuged at 5000 g for 20 minutes, the precipitate was discarded, and the supernatant was filtered through a 0.22 μm ultrafiltration membrane. The solution was then dialyzed in deionized water for 48 hours using a dialysis bag with a molecular weight cutoff of 500 Da, followed by vacuum concentration. Finally, the concentrated solution was lyophilized to obtain fluorescent CDs powder. A portion of the CDs powder was dissolved in deionized water and sonicated for 15 minutes to obtain a CDs aqueous solution with a concentration of 1 mg / mL. This solution was stored at 4°C for subsequent analysis.

[0050] Example 2: Preparation of fructose-derived glycosylated antibacterial carbon dots

[0051] 0.5 g of fructose and 0.5 g of BSA were mixed and dissolved in 20 mL of deionized water. The reaction solution was then placed in an autoclave and sealed using a PTFE-lined hydrothermal reactor. The reactor was then placed in a muffle furnace and reacted at 180 °C for 12 hours. After the reaction, the mixture was allowed to cool naturally to room temperature, yielding a brownish-black product. The reaction solution was then centrifuged at 5000 g for 20 minutes, the precipitate was discarded, and the supernatant was filtered through a 0.22 μm ultrafiltration membrane. The supernatant was then dialyzed in deionized water for 48 hours using a dialysis bag with a molecular weight cutoff of 500 Da, followed by vacuum concentration. Finally, the vacuum concentrate was lyophilized to obtain fluorescent CDs powder. A portion of the CDs powder was dissolved in deionized water and sonicated for 10 minutes to obtain a CDs aqueous solution with a concentration of 1 mg / mL. This solution was stored at 4 °C for subsequent analysis.

[0052] Example 3: Experimental Analysis and Testing

[0053] (1) Preparation process of glycosylated blue fluorescent antibacterial carbon dots

[0054] A typical characteristic of the glycosylated carbon dot preparation process in this invention is a burnt, bitter taste, somewhat similar to the aroma of strong coffee. Furthermore, the resulting freeze-dried powder is pale yellow or dark yellow, and also has a pungent odor. Figure 1 ).

[0055] (2) Appearance of glycosylated carbon dots under sunlight and ultraviolet light.

[0056] The experimental method was as follows: Glycosylated carbon dots from glucose source, prepared at 180℃ for 6 hours, were diluted at different gradients to achieve concentrations of 5.3400, 4.8060, 4.0050, 2.6700, 1.3350, 0.2670, and 0.0534 mg / mL, with deionized water as a control. Similarly, glycosylated carbon dots from fructose source, prepared at 180℃ for 12 hours, were diluted at different gradients to achieve concentrations of 0.9500, 0.8550, 0.7125, 0.4750, 0.2375, 0.0475, and 0.0095 mg / mL, with deionized water as a control.

[0057] Figure 2 (A) is an image of the appearance of the glycosylated carbon dots of glucose source described in Example 1 at 180℃ for 6h under sunlight and 395nm ultraviolet light.

[0058] Figure 2 (B) Images of the appearance of the fructose source glycosylated carbon dots described in Example 2 at 180℃ for 12h under sunlight and 395nm ultraviolet light.

[0059] The results show that both types of glycosylated carbon dots emit blue fluorescence, and the intensity of blue fluorescence weakens as the concentration decreases (5.34 mg / mL to 0.0534 mg / mL) and (0.95 mg / mL to 0.0095 mg / mL).

[0060] (3) UV-Vis absorption spectroscopy characterization of glycosylated carbon dots

[0061] The experimental method was as follows: A 1 mg / mL aqueous solution of fluorescent CDs was subjected to spectral scanning in the range of 200–700 nm using a UV-Vis spectrophotometer. Distilled water was used as a blank control.

[0062] The results show that both glycosylated CDs in Examples 1 and 2 exhibit significant light absorption in the near-ultraviolet region (230-270 nm; 270-320 nm). This demonstrates the characteristics of carbon dots, namely the π-π* electronic transitions of C=C on aromatic groups (core region) and the n-π* electronic transitions of C=O or other surface groups (edge ​​region). Figure 3 ).

[0063] (4) Fluorescence spectral characterization of glycosylated carbon dots

[0064] The experimental method was as follows: Fluorescent CDs were prepared into a 1 mg / mL solution, and a full-wavelength scan was performed using a fluorescence spectrometer to determine their fluorescence spectral characteristics and observe the change in emission wavelength with excitation wavelength. The fluorescence spectral measurement conditions were: scan speed: 1500 nm / min; time delay: 0.0 s; excitation slit width: 5.0 nm; emission slit width: 5.0 nm; voltage: 400 V; excitation wavelength: 280–450 nm; emission wavelength: 300–600 nm; acquisition wavelength step: 10 nm.

[0065] The results show that both glycosylated carbon dots (CDs) in Examples 1 and 2 exhibited two emission peaks in the 400-500 nm emission band. Furthermore, the maximum fluorescence intensity gradually decreased (from high to low) as the concentration of glycosylated carbon dots decreased. At the same concentration of glycosylated carbon dots, the maximum fluorescence intensity of the fructose-derived glycosylated carbon dots at 180℃ for 12 h was stronger than that of the glucose-derived glycosylated carbon dots at 180℃ for 6 h. Figure 4 ).

[0066] (5) CIE characterization of glycosylated carbon dots

[0067] The experimental method was as follows: Glycosylated carbon dots from glucose source, obtained at 180℃ for 6 hours, were diluted at different gradients to achieve concentrations of 5.3400, 4.8060, 4.0050, 2.6700, 1.3350, 0.2670, and 0.0534 mg / mL. Similarly, glycosylated carbon dots from fructose source, obtained at 180℃ for 12 hours, were diluted at different gradients to achieve concentrations of 0.9500, 0.8550, 0.7125, 0.4750, 0.2375, 0.0475, and 0.0095 mg / mL. The spectral data were imported into Origin software, and CIE visualization color maps were created using the CIE package.

[0068] The CIE colorimetric diagram is a three-dimensional chart drawn based on the characteristics of human color perception, used to describe the position of colors in color space. The colorimetric diagram mainly describes the coordinate position of colors in the red, green, and blue directions. It is a cube-shaped graphic, where the three coordinates represent the brightness values ​​of the color in the red, green, and blue directions, respectively, with the center point being gray. The results show that, under fluorescent light, the two types of glycosylated carbon dots in Examples 1 and 2 are both located in the blue region of the color space, thus proving that the glycosylated carbon dots are blue fluorescent carbon dots (…). Figure 5 ).

[0069] (6) Antibacterial characterization of glycosylated carbon dots

[0070] The experimental method was as follows: 20g of lactic acid bacteria culture medium was weighed into a 1000mL Erlenmeyer flask, and 800mL of ultrapure water was added. The solution was dissolved in a constant temperature water bath at 100℃, stirred until clear and non-turbid, and then cooled to room temperature. The medium was then sterilized in an autoclave at 0.1MPa and 121℃ for 30min. After cooling to room temperature, it was set aside for later use. 15mL–20mL of the culture medium was dispensed into sterile petri dishes and allowed to solidify. Sterile water was used as the solvent in the preparation of the glycosylated carbon dots (CDs) solution to avoid interference from other microorganisms. 0.05 mg / mL of glycosylated carbon dots was added to the culture medium, with sterile water as a control. The culture was incubated at 37℃ for 48 hours.

[0071] The results show that the culture medium containing 0.05 mg / mL of glycosylated carbon dots had fewer colonies compared to the culture medium without glycosylated carbon dots, thus proving that the prepared blue glycosylated fluorescent carbon dots have antibacterial properties. Figure 6 ).

[0072] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of blue fluorescent antibacterial carbon dots based on glycosylation reaction in the preparation of antibacterial additives for meat products, characterized in that, The preparation method of the blue fluorescent antibacterial carbon dots includes the following steps: (A) Mix carbohydrate compounds with bovine serum albumin and place them in a hydrothermal reactor. React the mixture at 120℃~180℃ for 6-12 hours. (B) Dissolve and centrifuge the intermediate slurry prepared in step (A), take the supernatant, dialyze, and freeze dry to obtain a yellowish-brown or dark yellow powder with a pungent odor, which is a blue fluorescent antibacterial carbon dot powder. The hydrothermal reaction in step (A) also includes controlling the pH value between 4 and 7. The hydrothermal reaction in step (A) is a reaction in which carbohydrate compounds are mixed with water at a mass-volume ratio of 1 g: 10~40 mL. The carbohydrate compounds mentioned in step (A) are glucose and / or fructose.

2. The application according to claim 1, characterized in that, In step (A), the mass ratio of carbohydrates to bovine serum albumin is 1:

1.

3. The application according to claim 1, characterized in that, The intermediate in step (B) is a dark brown crude extract with a strong coffee roast aroma.

4. The application according to claim 1, characterized in that, The centrifugation conditions described in step (B) are 5000. g , 20min.

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