Use of glucosamine in the degradation of tetracycline antibiotics in water bodies

Abstract

The application relates to application of glucosamine in degradation of tetracycline antibiotics in water bodies, and belongs to the technical field of removal and degradation of antibiotic residues. The application specifically discloses a method for degrading tetracycline antibiotics (TCs) in water bodies by using a natural compound glucosamine (GlcN), which can reduce the pollution risk of tetracycline antibiotics to the environment while ensuring the degradation of tetracycline antibiotics in water bodies, and can also widen the application range of glucosamine (GlcN). In addition, the glucosamine (GlcN) used in the application is derived from chitin and chitosan in crustacean shells and is obtained through natural extraction or microbial fermentation, has the characteristic of being renewable, makes the application method more sustainable, and has good applicability and high removal capacity, has a degradation effect on various tetracycline antibiotics, and has a better degradation effect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of removal and degradation of antibiotic residues, and relates to application of glucosamine in degradation of tetracycline antibiotics in water bodies. BACKGROUND

[0002] Since the discovery of penicillin in the 20th century, the introduction of antibiotics has played an important role in clinical treatment, which significantly reduces the mortality rate of pathogenic bacterial infection and the risk of various infections, and makes a great contribution to alleviating the burden of public health systems. In the past few decades, antibiotics have been widely used to treat or prevent microbial infection diseases in humans and animals, in addition, in agriculture and aquaculture, antibiotics are also commonly used as growth promoters for livestock and fish, and for the prevention and treatment of bacterial diseases. However, among all the antibiotics used by humans and livestock, 30-90% of the antibiotics are excreted into the environment in the form of a mixture of drug prototypes and metabolites through urine and feces. Another major source of antibiotic residues is aquaculture, which accounts for about 71% of global aquaculture production in China. According to literature reports, 70-80% of the antibiotics used in fish farms eventually enter the environment. The used antibiotics are released into the environment through different channels, such as various water bodies, agricultural activities, surface runoff, and waste discharge from animal facilities. With the widespread use of antibiotics, a large amount of antibiotics are left in rivers, oceans, municipal sewage, groundwater, drinking water, soil, and vegetables. These residues may have negative effects on the environment, ecosystems, and human health, including antibiotic residues in aquaculture products, the occurrence of bacterial drug resistance, and the disruption of water ecological balance.

[0003] Tetracycline antibiotics (TCs) have been widely used in aquaculture and animal husbandry due to their low price, easy use, and fewer side effects. The most commonly used tetracycline drugs include tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), and doxycycline (DOX) used in veterinary clinics, and tigecycline (TIG) and minocycline (MNO) used in human medicine. The widespread use of TCs in the treatment and prevention of human diseases and in aquaculture and agriculture has led to the continuous influx and accumulation of TCs, which will cause damage to the safety and diversity of organisms in the environment and pose a serious threat to human safety. Therefore, it is of great research significance to degrade such antibiotic residues in environmental water bodies, especially using non-toxic compounds existing in nature to promote the degradation of antibiotic residues, which not only can reduce the negative impact of antibiotics on the environment and ecosystems, but also helps to protect public health and promote sustainable development. Therefore, it is crucial to explore suitable materials, dosage, and temperature to effectively solve the problem of antibiotic residues in the environment.

[0004] Current research on antibiotic degradation mainly includes the exploration and optimization of adsorption, chemical, and biological methods, such as filtration, coagulation, flocculation, sedimentation, advanced oxidation processes (AOPs), adsorption, membrane processes, biodegradation, and combined methods. Among them, ozonation, Fenton / Photo-Fenton, and semiconductor photocatalysis are the most tested methods. However, existing antibiotic removal methods have both advantages and disadvantages. For example, physical and chemical methods for degrading antibiotics are generally effective, but these methods have strict operating requirements during the treatment process, ultimately leading to difficulty in achieving ideal degradation results. However, biological degradation is an eco-friendly method, but its low efficiency and the risk of horizontal transfer of drug-resistant genes in nature limit its further application. Therefore, it is crucial to develop more efficient, environmentally friendly, sustainable, and low-risk methods for antibiotic degradation, which deserves human attention.

[0005] Glucosamine (GlcN) is a naturally occurring compound in which the hydroxyl group of glucose is replaced by an amino group. It is also an important component of polysaccharides chitin and chitosan and an important compound required for chondrocyte composition. In nature, GlcN is widely present in the tissues of animals and plants, especially in cartilage, connective tissue, and the shells of crustaceans. Currently, GlcN has been widely used in the food, cosmetics, health care, and pharmaceutical industries. For example, GlcN can be used as a nutritional supplement in food, and it is the fourth most commonly used dietary supplement globally. In addition to nutritional supplements, GlcN is also used as an additive in food and sports drinks, has the benefit of improving joint health, and is used in cosmetics to improve skin moisture. In addition, in medicine, GlcN is commonly used for the treatment of osteoarthritis, which has the function of reducing joint pain and delaying the progression of osteoarthritis structure. However, there is no report on the role of GlcN in antibiotic degradation.

[0006] The inventors of the present application found in their previous research that the presence of GlcN can significantly reduce the antibacterial activity of the tetracycline antibiotic Tigecycline. Further exploration found that it can accelerate the degradation of tetracycline antibiotics represented by tetracycline. However, there is little understanding of the degradation efficiency, mechanism, properties of degradation products, and degradation efficiency and influencing factors of GlcN in antibiotic degradation. Research in this field is still in its infancy and needs further in-depth research and exploration. There are still many unknown factors about the potential application and effect of GlcN in antibiotic degradation. More scientific research will help to reveal the mysteries of this field and provide new ideas and directions for the development of environmentally friendly antibiotic degradation methods.

[0007] Therefore, the degradation of residual antibiotics in water bodies is of significant research importance. By finding and developing effective degradation methods, the potential harm of antibiotics to water bodies can be reduced, and the health of ecosystems can be maintained. Of particular importance is the growing focus on using naturally occurring, non-toxic compounds to promote the degradation of residual antibiotics. This approach not only reduces the risk of environmental pollution but also possesses potential sustainability and eco-friendliness. Summary of the Invention

[0008] In view of this, the object of the present invention is to provide the application of glucosamine in the degradation of tetracycline antibiotics.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] 1. Application of glucosamine in the degradation of tetracycline antibiotics in water.

[0011] Preferably, the concentration of glucosamine (GlcN) in the application is ≥1mM.

[0012] Preferably, the tetracycline antibiotics include any one or more of tetracycline, tigecycline, minocycline, doxycycline, chlortetracycline, or oxytetracycline.

[0013] Preferably, the concentration of tetracycline antibiotics used in the application is 16–256 μg / mL.

[0014] Preferably, the water body includes ultrapure water, tap water, river water, aquaculture water, and wastewater treatment plant water.

[0015] The beneficial effects of this invention are as follows: This invention discloses the application of glucosamine in the degradation of tetracycline antibiotics in water. This application is a green, environmentally friendly, pollution-free, simple, and highly efficient method for catalytic degradation of tetracycline antibiotics in water using the natural compound glucosamine (GlcN). Specifically, it utilizes the natural compound glucosamine (GlcN) to reduce the environmental pollution risk of tetracycline antibiotics while ensuring the degradation of tetracycline antibiotics (TCs) in water, thus broadening the application scope of glucosamine (GlcN). Furthermore, since the glucosamine (GlcN) used in this invention is derived from chitin and chitosan in the shells of crustaceans and obtained through natural extraction or microbial fermentation, it has renewable characteristics, making the application of this invention for degrading tetracycline antibiotics in water more sustainable. At the same time, the glucosamine (GlcN) used has good applicability and high removal efficiency, exhibiting degradation effects on various tetracycline antibiotics with excellent degradation results.

[0016] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0017] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0018] Figure 1 The effect of glucosamine (GlcN) on the diameter of the inhibition zone of six tetracycline antibiotics, where A to F are TC (tetracycline), TIG (tigecycline), MNO (minocycline), OTC (oxytetracycline), CTC (chlortetracycline), and DOX (doxycycline).

[0019] Figure 2 The effect of different concentrations of glucosamine (GlcN) on the degradation efficiency of six tetracycline antibiotics within 24 hours at 37℃ was investigated. Among them, a to f are tetracycline, tigecycline, minocycline, doxycycline, chlortetracycline, and oxytetracycline.

[0020] Figure 3 A heatmap of tetracycline (TC) degradation efficiency (TC degradation efficiency after co-incubation of 16–256 μg / mL tetracycline (TC) with 0–200 mM glucosamine (GLcN) for 24 h);

[0021] Figure 4 The effects of glucosamine (GlcN) on the degradation of tetracycline (TC) at different pH levels;

[0022] Figure 5 The degradation of tetracycline (TC) by glucosamine (GlcN) at different temperatures;

[0023] Figure 6 The degradation efficiency of tetracycline (TC) at 20℃ under different reaction times and different glucosamine concentrations;

[0024] Figure 7The inhibition zone diameter data (a) and the intuitive display of experimental results (b) are obtained from the investigation of the antibacterial activity of the degradation products of six different tetracycline antibiotics after accelerated degradation by GlcN against different bacteria. Among them, TCs represent different tetracycline antibiotics, DP-TCs represent the degradation products of different tetracycline antibiotics after complete degradation by glucosamine (GlcN), Blank represents the ultrapure water blank group, and Klebsiella pneumoniae, E. coli, S. aureus and Salmonella represent the tested strains such as Klebsiella pneumoniae (KP2229), Escherichia coli (ATCC25922), Staphylococcus aureus (CMCC(B)26003) and Salmonella (CMCC(B)50071), respectively.

[0025] Figure 8 The results of weight monitoring of mice before and after gavage administration of the degradation products of the blank control group (PBS) and six tetracycline drugs after accelerated degradation by glucosamine (GlcN);

[0026] Figure 9 Tissue epigraphs of mice in acute toxicity experiments of degradation products of six tetracycline antibiotics after accelerated degradation by glucosamine (GlcN) in the blank control group (PBS) and mice in the control group.

[0027] Figure 10 Histopathological sections of mice in an acute toxicity experiment of the degradation products of six tetracycline antibiotics after accelerated degradation by glucosamine (GlcN) were collected from the blank control group (PBS) and mice.

[0028] Figure 11 Electron paramagnetic resonance (EPR) spectrum of the TC degradation system;

[0029] Figure 12 The effect of different compounds on the degradation efficiency of tetracycline (TC) is shown in Figures a through f, where a–f represent the degradation rates of tetracycline (TC) within 24 hours after the addition of glucosamine (GlcN), glucosamine hydrochloride, glucosamine hydrochloride desalted, glucose, N-acetylglucosamine, and N-methyl-D-glucosamine, respectively. “+” indicates the addition of the corresponding compound and “-” indicates the absence of the corresponding compound.

[0030] Figure 13 The degradation efficiency of six tetracycline antibiotics was determined by adding glucosamine (GlcN) to five different water bodies, where TCs control represents the degradation rate of TCs without the addition of glucosamine (GlcN). Detailed Implementation

[0031] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0032] The main drugs and their sources in the following examples are as follows: Tigecycline was purchased from Tianjin Xiens Biochemical Technology Co., Ltd.; Glucosamine, Chlortetracycline Hydrochloride and Doxycycline Hydrochloride were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Oxytetracycline was purchased from Shanghai Aladdin Reagent Co., Ltd.; Tetracycline was purchased from Shanghai Mairui Chemical Technology Co., Ltd.; Minocycline Hydrochloride was purchased from Hefei Bomei Biotechnology Co., Ltd.

[0033] The main instrument models in the following examples are: Mettler Ag135 electronic balance and Agilent 1200 series high performance liquid chromatograph.

[0034] Example 1

[0035] Glucosamine accelerates the degradation of tetracycline antibiotics in aqueous solution, and the specific process is as follows:

[0036] 1. Drilling method for drug sensitivity testing

[0037] (1) Pour the autoclaved LB agar into a petri dish and let it solidify for later use;

[0038] (2) Tetracycline (TC) was dissolved in ultrapure water to prepare a tetracycline antibiotic solution (TCControl) with a concentration of 16 μg / mL. Tetracycline (TC) and glucosamine (GlcN) were dissolved in ultrapure water to obtain a mixed solution (TC+GlcN) with a tetracycline concentration of 16 μg / mL and a glucosamine concentration of 10 mM. Ultrapure water was used as the blank group. The solution was incubated at 37°C in the dark for 24 h to obtain three samples of the tetracycline sample group (TC Control, TC+GlcN and Blank).

[0039] (3) The laboratory-preserved standard strain of Escherichia coli ATCC25922 was used as the test strain. The ATCC25922 bacterial solution was cultured until the McFarland turbidity was 0.5 and then evenly spread on LB agar plates. Oxford cups were used to make holes (hole diameter was 0.9 cm) to form agar medium wells.

[0040] (4) Subsequently, the three samples obtained from the incubated tetracycline sample group in step (2) were added to the wells of agar medium at a dose of 100 μL per well, and then incubated overnight at 37°C for 20 h. After the incubation was completed, the samples were removed and the diameter of the inhibition zone of each sample was measured. The results are as follows: Figure 1 As shown in Figure A.

[0041] (5) Similarly, the five tetracycline antibiotics—tigecycline (TIG), minocycline (MNO), oxytetracycline (OTC), chlortetracycline (CTC), and doxycycline (DOX)—were subjected to the same steps (1) to (4) as described above. The diameter test results of the inhibition zones formed by the samples of each tetracycline antibiotic are as follows: Figure 1 As shown in B, C, D, E, and F.

[0042] from Figure 1 It can be seen that the antibacterial activity of all six tetracycline antibiotics (tetracycline (TC), tigecycline (TIG), minocycline (MNO), oxytetracycline (OTC), chlortetracycline (CTC), and doxycycline (DOX)) was significantly reduced after the addition of glucosamine (GlcN). Specifically, tetracycline, minocycline, oxytetracycline, doxycycline, and chlortetracycline completely lost their antibacterial activity after the addition of glucosamine (GlcN). Tigecycline, as a new generation tetracycline, showed significantly higher antibacterial efficacy than the other five drugs, possibly because it still retains some antibacterial activity against *Escherichia coli* ATCC25922 after 24-hour degradation. Compared to the six tetracycline antibiotics (TCs) alone, the diameter of the inhibition zone of tetracycline, minocycline, oxytetracycline, doxycycline, chlortetracycline, and tigecycline decreased by 0.8, 0.8, 0.7, 0.9, 1, and 0.6 cm, respectively, after the addition of glucosamine (GlcN). The above results indicate that the presence of glucosamine (GlcN) does indeed accelerate the degradation of tetracycline drugs.

[0043] 2. High-performance liquid chromatography (HPLC) for the detection of tetracycline drug degradation

[0044] (1) Tetracycline (TC) was dissolved in ultrapure water to prepare a tetracycline (TC) solution with a concentration of 16 μg / mL (TC-GlcN). 0mM Tetracycline (TC) and glucosamine (GlcN) were dissolved in ultrapure water to prepare tetracycline mixed solutions of different concentrations. These included tetracycline mixed solution I (TC-GlcN) containing 16 μg / mL tetracycline (TC) and 0.1 mM glucosamine (GlcN). 0.1mM Tetracycline mixed solution II (TC-GlcN) containing 16 μg / mL tetracycline (TC) and 1.0 mM glucosamine (GlcN) 1.0mMTetracycline mixed solution III (TC-GlcN) containing 16 μg / mL tetracycline (TC) and 5 mM glucosamine (GlcN) 5mM A tetracycline mixed solution IV (TC-GlcN) containing 16 μg / mL tetracycline (TC) and 10 mM glucosamine (GlcN) 10mM ).

[0045] (2) Replace the tetracycline (TC) in step (1) with tigecycline (TIG) to obtain a tigecycline (TIG) solution with a concentration of 16 μg / mL (TIG-GlcN). 0mM Tigecycline mixed solution I (TIG-GlcN) containing 16 μg / mL tigecycline (TIG) and 0.1 mM glucosamine (GlcN) 0.1mM Tigecycline mixed solution II (TIG-GlcN) containing 16 μg / mL tigecycline (TIG) and 1.0 mM glucosamine (GlcN) 1.0mM Tigecycline mixed solution III (TIG-GlcN) containing 16 μg / mL tigecycline (TIG) and 5 mM glucosamine (GlcN) 5mM Tigecycline mixed solution IV (TIG-GlcN) containing 16 μg / mL tigecycline (TIG) and 10 mM glucosamine (GlcN) 10mM ).

[0046] (3) Replace the tetracycline (TC) in step (1) with minocycline (MNO) to obtain a minocycline (MNO) solution with a concentration of 16 μg / mL (MNO-GlcN). 0mM Minocycline mixed solution I (MNO-GlcN) containing 16 μg / mL minocycline (MNO) and 0.1 mM glucosamine (GlcN) 0.1mM Minocycline mixed solution II (MNO-GlcN) containing 16 μg / mL minocycline (MNO) and 1.0 mM glucosamine (GlcN) 1.0mM Minocycline mixed solution III (MNO-GlcN) containing 16 μg / mL minocycline (MNO) and 5 mM glucosamine (GlcN) 5mM Minocycline mixed solution IV (MNO-GlcN) containing 16 μg / mL minocycline (MNO) and 10 mM glucosamine (GlcN) 10mM ).

[0047] (4) Replace tetracycline (TC) in step (1) with doxycycline (DOX) to obtain a doxycycline (DOX) solution with a concentration of 16 μg / mL (DOX-GlcN). 0mM), Doxycycline mixed solution I (DOX-GlcN) containing 16 μg / mL doxycycline (DOX) and 0.1 mM glucosamine (GlcN) 0.1mM Doxycycline mixed solution II (DOX-GlcN) containing 16 μg / mL doxycycline (DOX) and 1.0 mM glucosamine (GlcN) 1.0mM ), Doxycycline mixed solution III (DOX-GlcN) containing 16 μg / mL doxycycline (DOX) and 5 mM glucosamine (GlcN) 5mM ), Doxycycline mixed solution IV (DOX-GlcN) containing 16 μg / mL doxycycline (DOX) and 10 mM glucosamine (GlcN) 10mM ).

[0048] (5) Replace tetracycline (TC) in step (1) with chlortetracycline (CTC) to obtain a chlortetracycline (CTC) solution with a concentration of 16 μg / mL (CTC-GlcN). 0mM A chlortetracycline mixed solution I (CTC-GlcN) containing 16 μg / mL chlortetracycline (CTC) and 0.1 mM glucosamine (GlcN) 0.1mM A chlortetracycline mixed solution II (CTC-GlcN) containing 16 μg / mL chlortetracycline (CTC) and 1.0 mM glucosamine (GlcN) 1.0mM A chlortetracycline mixed solution III (CTC-GlcN) containing 16 μg / mL chlortetracycline (CTC) and 5 mM glucosamine (GlcN) 5mM A chlortetracycline mixed solution IV (CTC-GlcN) containing 16 μg / mL chlortetracycline (CTC) and 10 mM glucosamine (GlcN) 10mM ).

[0049] (6) Replace tetracycline (TC) in step (1) with oxytetracycline (OTC) to obtain an oxytetracycline (OTC) solution with a concentration of 16 μg / mL (OTC-GlcN). 0mM Oxytetracycline mixed solution I (OTC-GlcN) containing 16 μg / mL oxytetracycline (OTC) and 0.1 mM glucosamine (GlcN) 0.1mM Oxytetracycline mixed solution II (OTC-GlcN) containing 16 μg / mL oxytetracycline (OTC) and 1.0 mM glucosamine (GlcN) 1.0mM Oxytetracycline mixed solution III (OTC-GlcN) containing 16 μg / mL oxytetracycline (OTC) and 5 mM glucosamine (GlcN) 5mM ), Oxytetracycline mixed solution IV (OTC-GlcN) containing 16 μg / mL oxytetracycline (OTC) and 10 mM glucosamine (GlcN)10mM ).

[0050] The different solutions prepared from the above six tetracycline antibiotics were analyzed by high-performance liquid chromatography (HPLC). The specific methods for analysis are shown in Table 1 below, and the detection results are as follows. Figure 2 As shown, a to f are tetracycline, tigecycline, minocycline, doxycycline, chlortetracycline, and oxytetracycline.

[0051] Table 1. HPLC methods for analyzing solutions prepared from different tetracycline antibiotics.

[0052]

[0053]

[0054] The specific chromatographic conditions used also included: AQ-C18 column (250×4.6mm 5μm, 150×4.6mm 5μm); column temperature of 40℃, flow rate of 1mL / min, and injection volume of 25μL.

[0055] Combining bacterial drug susceptibility testing and high-performance liquid chromatography (HPLC) results, it was confirmed that the diameter of the inhibition zone of tetracycline antibiotics was significantly reduced in the presence of glucosamine. Compared with drug solutions alone, multiple chromatographic peaks of TC degradation products were detected in the presence of GlcN. Figure 2 It can be seen that there is a dose-dependent relationship between the degradation efficiency of the six tetracycline antibiotics and the concentration of glucosamine (GlcN). The addition of 10 mM glucosamine (GlcN) resulted in the highest degradation efficiency of minocycline in the short time (HPLC method time) (Figure 2c). Within 24 hours, the degradation percentage of all six tetracycline antibiotics continued to increase.

[0056] Similarly, tetracycline (TC) solutions of different masses were dissolved in ultrapure water to prepare concentrations of 16 μg / mL, 64 μg / mL, 128 μg / mL, and 258 μg / mL, respectively. These tetracycline solutions of different concentrations were then divided into eight equal portions, and different masses of glucosamine were added to each portion to achieve final glucosamine concentrations of 0 mM, 0.1 mM, 1.0 mM, 5.0 mM, 10 mM, 50 mM, 100 mM, and 150 mM, respectively. These solutions, containing different concentrations of tetracycline and glucosamine, were then analyzed by high-performance liquid chromatography (HPLC). The resulting thermograms of tetracycline (TC) degradation efficiency are shown below. Figure 3 As shown (a heatmap is a data visualization technique used to show the distribution of intensity or values ​​in matrix data. Through color gradients or contrasts, heatmaps can visually reveal hidden patterns, trends, and anomalies in the data). FromFigure 3 It can be seen that the degradation rate of tetracycline is positively correlated with the concentration of glucosamine and negatively correlated with its own concentration. When the concentration of tetracycline increases, increasing the amount of glucosamine can still further degrade tetracycline, and even achieve 100% degradation.

[0057] Tetracycline (TC) was dissolved in ultrapure water to form a tetracycline (TC) solution with a concentration of 16 μg / mL, which served as the GlcN- group. This solution was divided into six equal parts, and the pH of five of these parts was adjusted to 3, 5, 7, 9, and 11, respectively. The unadjusted mixed solution served as the control group (TC control). Tetracycline (TC) and glucosamine (GlcN) were dissolved in ultrapure water to form a mixed solution with a tetracycline (TC) concentration of 16 μg / mL and a glucosamine (GlcN) concentration of 10 mM, which served as the GlcN+ group. This solution was also divided into six equal parts, and the pH of five of these parts was adjusted to 3, 5, 7, 9, and 11, respectively. The unadjusted mixed solution served as the control group (TC control). High-performance liquid chromatography (HPLC) analysis was performed, and the results are as follows: Figure 4 As shown, GlcN+ indicates the addition of glucosamine (GlcN), and GlcN- indicates the absence of glucosamine (GlcN). From Figure 4 It can be seen that, compared with the ultrapure water system, there is no significant difference in the degradation effect of glucosamine on tetracycline under pH conditions of 5 and 7; while under pH conditions of 3, 9 and 11, the degradation effect of GlcN on TC is significantly reduced, but all are significantly higher than the TC degradation efficiency without the addition of GlcN under the same pH conditions.

[0058] Tetracycline (TC) and glucosamine (GlcN) were dissolved in ultrapure water to form a mixed solution with a tetracycline (TC) concentration of 16 μg / mL and a glucosamine (GlcN) concentration of 10 mM. This solution was divided into six equal portions, and high-performance liquid chromatography (HPLC) was performed at 70℃, 50℃, 37℃, 25℃, 4℃, and -20℃, respectively. The results are as follows: Figure 5 As shown. From Figure 5 It can be seen that under the temperature conditions of -20 to 70℃, the efficiency of glucosamine (GlcN) in promoting the degradation of tetracycline (TC) gradually increases with the increase of temperature, indicating that the degradation rate of TC is also positively correlated with temperature. When the temperature exceeds 50℃, 10mM glucosamine can completely degrade 16μg / mL of TC within 4h; at 37℃, the degradation rate of TC can also reach 100% within 24h with the extension of time.

[0059] Tetracycline (TC) was dissolved in ultrapure water to form a tetracycline (TC) solution with a concentration of 16 μg / mL. This solution was divided into eight equal portions, and different concentrations of glucosamine (GlcN) were added to each portion to form mixed solutions containing glucosamine (GlcN) at concentrations of 0 mM, 10 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, and 300 mM, respectively. High-performance liquid chromatography (HPLC) analysis was performed after 24 h, 36 h, and 48 h of reaction. The results are shown below. Figure 6 As shown. By Figure 6 It was found that, under 20℃ conditions, the degradation rate of TC was positively correlated with both reaction time and GlcN dosage. For the same reaction time, the higher the concentration of added glucosamine (GlcN), the higher the degradation rate of tetracycline (TC). Similarly, with the same concentration of glucosamine (GlcN), the longer the reaction time, the higher the degradation rate of tetracycline (TC). With 10mM glucosamine, the degradation rate of TC after 24 hours was only 9.53±3.02%, but increased to 86.88±1.61% after 48 hours. However, when the GlcN concentration increased to 300mM, the degradation efficiency of TC after 24 hours significantly improved to 82.11±1.68%, and reached 98.44±0.16% after 48 hours. The above results indicate that even when the reaction temperature decreases, the degradation of residual TCs in the water can still be achieved efficiently over time, or the rapid degradation of TCs can be achieved by increasing the dosage of GlcN.

[0060] Example 2

[0061] Safety study of glucosamine for removing tetracycline antibiotics from water

[0062] The incomplete absorption of antibiotics, resulting in their excretion as unchanged drugs, undoubtedly puts antibiotic stress on environmental microorganisms, potentially promoting the emergence of drug-resistant bacteria. Therefore, examining the antibacterial activity and toxicity of their degradation products is of great significance. The results showed that the degradation products of tetracycline antibiotics accelerated by GlcN completely lost their antibacterial activity and did not exhibit acute toxicity in mice. The specific experimental procedure and results are as follows:

[0063] 1. Investigation of the antibacterial activity of tetracycline antibiotic degradation products

[0064] (1) Pour the autoclaved LB agar into a petri dish and let it solidify for later use;

[0065] (2) Different tetracycline antibiotics (TCs) (tigecycline (TIG), minocycline (MNO), tetracycline (TC), doxycycline (DOX), and chlortetracycline (CTC)) were dissolved in ultrapure water to obtain a tetracycline antibiotic solution with a concentration of 16 μg / mL. Different tetracycline antibiotics (TCs) (tigecycline (TIG), minocycline (MNO), tetracycline (TC), doxycycline (DOX), chlortetracycline (CTC), and oxytetracycline (OTC)) and glucosamine (GlcN) were dissolved in ultrapure water to obtain a mixed solution with a tetracycline antibiotic concentration of 16 μg / mL and a glucosamine concentration of 50 mM. The solutions were incubated at 37°C in the dark for 48 h to obtain the test solution after incubation.

[0066] (3) Klebsiella pneumoniae (KP2229), Escherichia coli ATCC25922, Staphylococcus aureus CMCC(B)26003 and Salmonella CMCC(B)50071, which were prepared in the laboratory, were used as test strains. After the test strains were cultured to a McFarland turbidity of 0.5, they were evenly spread on LB agar plates and agar medium wells were formed by punching holes (0.9 cm in diameter) using Oxford cups.

[0067] (4) Then, the various solutions obtained in step (2) above were added to the wells of agar medium at a dose of 100uL per well, and then placed in an incubator at 37℃ for overnight culture for 20h. After the culture was completed, the samples were taken out and the diameter of the inhibition zone of each sample was measured.

[0068] Figure 7 The inhibition zone diameter data (a) and the intuitive display of experimental results (b) are obtained from the investigation of the antibacterial activity of the degradation products of six different tetracycline antibiotics after accelerated degradation by GlcN against different bacteria. TCs represent different tetracycline antibiotics, DP-TCs represent the degradation products of different tetracycline antibiotics after complete degradation by glucosamine (GlcN), Blank represents the ultrapure water blank group, and Klebsiella pneumoniae, E. coli, S. aureus, and Salmonella represent the tested strains such as Klebsiella pneumoniae (KP2229), Escherichia coli (ATCC25922), Staphylococcus aureus (CMCC(B)26003), and Salmonella (CMCC(B)50071), respectively. Figure 7The results show that after the six tetracycline antibiotics were completely degraded by glucosamine (GlcN), the degradation products no longer possessed antibacterial activity and showed no inhibitory effect on any of the four tested bacterial strains. The diameter of the inhibition zone was consistent with that of the blank control group. This indicates that using glucosamine (GlcN) for the removal of tetracycline antibiotics from water bodies is environmentally friendly, does not pose a risk of secondary environmental pollution, effectively reduces the antibiotic pressure on microorganisms from residual parent drugs, and can effectively slow down the process of drug-resistant bacteria development.

[0069] 2. Acute toxicity study of tetracycline antibiotic degradation products

[0070] Km mice aged 8-10 weeks and weighing approximately 18-20g were purchased and acclimatized to their environment for 5 days before the experiment. They were fasted for 12 hours before gavage. There were 7 groups, with 6 mice in each group: a blank control group (PBS), a group containing tigecycline (TIG), a group containing minocycline (MNO), a group containing tetracycline (TC), a group containing doxycycline (DOX), a group containing chlortetracycline (CTC), and a group containing oxytetracycline (OTC) drug degradation products (containing 500 mg / mL tetracycline antibiotics and 50 mM glucosamine). The mice were degraded at a dose of 500 mg / kg (incubated at 37°C for 48 hours to obtain high concentrations of TC degradation products after GlcN degradation) and then administered the drugs via gavage for three consecutive days. The mice's vital signs and mental state were closely monitored for 12 hours after each gavage. Ten days after the first gavage, the mice were dissected and their heart, liver, spleen, lung, and kidney tissues were collected for external observation. In each group, one mouse was randomly selected to have its heart, liver, spleen, lung, and kidney tissues stained with H&E for pathological study. The weight of the mice was monitored during the experiment.

[0071] Figure 8 The results show the body weight monitoring of mice before and after oral gavage administration of the blank control group (PBS) and the degradation products of six tetracycline antibiotics accelerated by GlcN. Figure 8 The results showed that in the acute toxicity test of the degradation products of six tetracycline antibiotics at a dose of 500 mg / kg, the weight change trend of the mice in the six experimental groups was basically consistent with that of the PBS control group. During the 15-day monitoring period, the weight of all seven groups of mice showed a gradual increasing trend, with consistent weight fluctuations during gavage. After gavage, the mice showed good mental state and did not exhibit a series of adverse symptoms such as lethargy, rapid heartbeat, ruffled fur, convulsions, and decreased appetite. These results indicate that the tetracycline antibiotic degradation products accelerated by GlcN did not cause any visible acute toxicity in mice.

[0072] Figure 9Tissue epiphyses of mice in acute toxicity experiments using GlcN-accelerated degradation products of the blank control group (PBS) and six tetracycline antibiotics. Figure 9 It was found that no significant visual tissue lesions were found in the heart, liver, spleen, lungs and kidneys of the 7 groups of mice.

[0073] Figure 10 Histopathological sections of mice subjected to acute toxicity experiments using the degradation products of six tetracycline antibiotics after accelerated degradation by glucosamine (GlcN), serving as a blank control group (PBS). Figure 10 It can be seen that, compared with the PBS control group, the histopathological results of the mice in the 6 experimental groups showed no significant differences or abnormalities between the two groups.

[0074] Example 3

[0075] 1. Investigation into the mechanism and efficiency of glucosamine (GlcN) in promoting the accelerated degradation of tetracycline drugs.

[0076] To detect the presence of free radicals, glucosamine (GlcN), glucosamine hydrochloride (GlcN·HCl), and desalted glucosamine hydrochloride (GlcNΔHCl) were added to a tetracycline (TC) solution to prepare a 190 μL mixed solution. The final concentrations of GlcN, GlcN·HCl, and GlcNΔHCl were all 10 mM, and the final concentration of TC was 16 μg / mL. 10 μL of 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO) was added as a spin trap to characterize the generation of ·OH radicals via electron spin resonance (EPR). The solution was immediately placed in the instrument after the addition of DMPO for EPR signal detection. Figure 11 The image shows the electron paramagnetic resonance (EPR) spectrum of the TC degradation system. Figure 11 It can be seen that in GlcN 10mM Hydroxyl radical (·OH) signals were detected in all solutions present, however in GlcN·HCl. 10mM The signal was not detected in the solution. After desalting, GlcNΔHCl was added. 10mM Hydroxyl radicals were also detected in the solution. This result is consistent with the HPLC results, indicating that glucosamine accelerates the degradation of tetracycline antibiotics, while glucosamine hydrochloride does not have this effect. Desalted glucosamine regains this tetracycline-promoting degradation effect. The above suggests that the tetracycline-promoting degradation effect of glucosamine (GlcN) may be due to the hydroxyl radicals present in its aqueous solution stimulating the active sites of tetracycline antibiotics, leading to a series of reactions such as oxidation and hydrolysis, thereby promoting the degradation of tetracycline antibiotics.

[0077] A series of compounds, including glucosamine (GlcN), glucosamine hydrochloride, glucosamine hydrochloride desalted from glucosamine hydrochloride, glucose, N-acetylglucosamine, and N-methyl-D-glucosamine, were added to a tetracycline (TC) solution at a concentration of 16 μg / mL, with a final concentration of 10 mM. The tetracycline (TC) degradation efficiency was measured before the addition of the compounds and at different time points after addition. The results are shown below. Figure 12 As shown in Table 2, a to f are the values ​​in the table.

[0078] Table 2. Degradation rate of tetracycline after adding different compounds.

[0079]

[0080] Figure 12 In the figures, a to f represent the degradation rates of tetracycline (TC) within 24 hours after the addition of glucosamine (GlcN), glucosamine hydrochloride, glucosamine hydrochloride desalted, glucose, N-acetylglucosamine, and N-methyl-D-glucosamine, respectively. "+" indicates that the corresponding compound was added to the TC solution, and "-" indicates that the corresponding compound was not added to the TC solution. Figure 12 The degradation rate of TC in the sample shows that only glucosamine (GlcN) capable of producing ·OH is present. Figure 12 desalting of glucosamine hydrochloride (a) and glucosamine hydrochloride (a) Figure 12 The product treated with c) exhibits a degradation effect on tetracycline, while the amino group in the glucosamine structure is replaced by a hydroxyl group. Figure 12 (d) or forms ionic bonds with other groups ( Figure 12 (b) or covalent bond ( Figure 12 When incubation occurs in steps e and f), the tetracycline degradation activity is lost. Specifically, glucosamine (GlcN) exhibits a near 100% degradation efficiency for tetracycline (TC) after 24 hours, while glucosamine hydrochloride, when co-incubated with tetracycline (TC) for 24 hours, only degrades TC by 5.70±1.03%, showing no significant difference compared to the tetracycline (TC) control group (2.93±3.35%). However, the tetracycline degradation activity of glucosamine hydrochloride can be restored after desalting treatment. This indicates that the degradation effect of glucosamine on tetracycline drugs is significantly related to its main cyclic structure and amino groups.

[0081] 2. Investigation on the efficiency of glucosamine in accelerating the degradation of tetracycline drugs in different environmental water bodies.

[0082] River water, tap water, wastewater, aquaculture water, and ultrapure water were collected to investigate the degradation-promoting effect of glucosamine (GlcN) on tetracycline antibiotics in different environmental water bodies. Tetracycline antibiotics (TCs) were used as a control group to explore the practical application value of glucosamine (GlcN). Different tetracycline antibiotics (TCs) and glucosamine (GlcN) were added to different water samples, with a final concentration of 16 μg / mL for the tetracycline antibiotics and 10 mM for glucosamine (GlcN). The samples were incubated at 37℃ for 24 h for degradation. After filtration through a 0.22 μm filter membrane, all samples were analyzed using the HPLC method described above. The degradation efficiencies of six tetracycline antibiotics after the addition of glucosamine (GlcN) in the five different water bodies are shown below. Figure 13 As shown, TCscontrol represents the degradation rate of TCs without the addition of glucosamine (GlcN). From Figure 13 It can be seen that the degradation efficiency of six tetracycline antibiotics in aquaculture water, river water, tap water, urban sewage and ultrapure water was significantly improved after the addition of glucosamine (GlcN), and the degradation rate of many drugs can reach 100%.

[0083] The above experiments demonstrate that glucosamine, as a tetracycline antibiotic degrader, can significantly improve the degradation efficiency of tetracycline drugs in aquatic environments. The degradation products have no antibacterial activity and exhibit good biocompatibility in mice. Based on this phenomenon, related applications are being developed, namely, by adding glucosamine to the aquatic environment or by composting tetracycline residues with chitin-rich chitin-rich waste, the residual tetracycline drugs are degraded by microbial decomposition using GlcN. Simultaneously, H&E staining results in mouse tissues show that there is no significant damage to the animals after accelerated degradation of tetracycline drugs by GlcN. Therefore, it can be inferred that GlcN can serve as a novel, highly efficient, low-energy-consumption, and environmentally friendly tetracycline drug degrader for application in the field of environmental antibiotic pollution treatment.

[0084] In summary, the natural product glucosamine can efficiently promote the degradation of tetracycline antibiotics in various water bodies, and the degradation products have the advantages of no antibacterial activity and no toxicity. The degradation mechanism may be related to the presence of hydroxyl radicals in the GlcN solution. Therefore, glucosamine (GlcN) can be used as a green, safe, efficient, and renewable novel tetracycline drug degrading agent in the field of environmental antibiotic pollution treatment.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The application of glucosamine in the degradation of tetracycline antibiotics in water, characterized by: Glucosamine promotes the degradation of tetracycline drugs due to the generation of hydroxyl radicals in their aqueous solutions.

2. The application according to claim 1, characterized in that, The concentration of glucosamine in the application is ≥1 mM.

3. The application according to claim 1, characterized in that, The tetracycline antibiotics include any one or more of tetracycline, tigecycline, minocycline, doxycycline, chlortetracycline, or oxytetracycline.

4. The application according to claim 1, characterized in that, The concentration of tetracycline antibiotics used in this application is 16~256 µg / mL.

5. The application according to claim 1, characterized in that, The water bodies mentioned include ultrapure water, tap water, river water, aquaculture water, and sewage treatment plant water.

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