Composite photocatalytic antibacterial agent and preparation method thereof

Abstract

The application discloses a composite photocatalytic bacteriostatic agent and a preparation method thereof, and belongs to the technical field of aquatic diseases. The emodin (Em) is loaded on sodium alginate (SA) to prepare the composite photocatalytic bacteriostatic agent Em / SA. Compared with the emodin alone, the composite photocatalytic bacteriostatic agent Em / SA can further improve the inactivation effect on Aeromonas hydrophila cells. The survival rate of the Aeromonas hydrophila with a bacterial concentration of 10 7 cfu / mL is reduced from 99.33% when the emodin is used alone to less than 21.65%. The preparation method of the photocatalytic bacteriostatic agent Em / SA is simple and green, the composite photocatalytic bacteriostatic agent Em / SA only contains the natural product emodin and sodium alginate, the reaction is carried out under light, no secondary pollution is caused to the environment, and the photoinactivation of the Aeromonas hydrophila can be efficiently realized under white light illumination for 30 min.

Description

Technical Field

[0001] This invention relates to a composite photocatalytic antibacterial agent and its preparation method, belonging to the field of aquatic disease technology. Background Technology

[0002] Aeromonas hydrophila is widely distributed in freshwater environments and is a major pathogen infecting fish, causing diseases such as furunculosis, ulceration, and hemorrhagic septicemia. These diseases are characterized by wide prevalence, long epidemic cycles, and high mortality rates, resulting in economic losses of tens of billions of yuan annually.

[0003] In aquaculture, antibiotics are commonly used to treat Aeromonas hydrophila infections. However, the overuse and even abuse of antibiotics not only pollute the environment but also induce antibiotic resistance. Therefore, developing a novel, green antibacterial agent with significant antibacterial effects is of great importance.

[0004] Emodin, a natural product of traditional Chinese medicine, possesses excellent optical properties due to its large π-conjugated anthraquinone structure. It can generate reactive oxygen species (ROS) under visible light irradiation, exhibiting advantages such as high efficiency, lack of drug resistance, and no side effects, making it suitable as a photocatalytic antibacterial agent. Existing patent CN115590062A discloses the use of emodin as a photocatalytic antibacterial agent for the inactivation of Escherichia coli and Staphylococcus aureus, but the inactivation effect varies for different bacterial species. Existing technologies do not disclose the preparation of composite photocatalytic antibacterial agents using emodin to further enhance its photocatalytic antibacterial effect, or its application in the inactivation of Aeromonas hydrophila. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a composite photocatalytic antibacterial agent, prepared by loading emodin (Em) into sodium alginate (SA). The composite photocatalytic antibacterial agent Em / SA can significantly enhance the photocatalytic antibacterial effect of emodin.

[0006] This invention provides a composite photocatalytic antibacterial agent, which is composed of sodium alginate and at least one of the following substances (a)-(b):

[0007] (a) Emodin as an active ingredient;

[0008] (b) Emodin derivatives with anthraquinone structures;

[0009] In one embodiment of the present invention, the emodin derivative includes aloe-emodin, emodin methyl ether, rhein, or rhein.

[0010] This invention also provides a method for preparing a composite photocatalytic antibacterial agent, the method comprising the following steps:

[0011] (1) Mix emodin or emodin derivatives with sodium alginate aqueous solution and stir;

[0012] (2) Mix the solution obtained in step (1) with the calcium chloride solution;

[0013] (3) Rinse the mixture obtained in step (2), pre-cool it, freeze dry it to obtain a composite photocatalytic antibacterial agent.

[0014] In one embodiment of the present invention, in step (1), the mass ratio of emodin to or the emodin derivative sodium alginate is 1:5 or more, or 1:(5-100), or 1:(5-20).

[0015] In one embodiment of the present invention, in step (1), the sodium alginate aqueous solution is obtained by mixing sodium alginate with water and stirring to dissolve it, and the concentration of the sodium alginate aqueous solution is 5-20 g / L, preferably 10 g / L.

[0016] In one embodiment of the present invention, the stirring time in step (1) is 6-10 hours, preferably 8 hours.

[0017] In one embodiment of the present invention, the mixing method in step (2) is to add the solution obtained in step (1) dropwise into the calcium chloride solution, or to add the solution obtained in step (1) dropwise into the calcium chloride solution at a rate of 5-20 mL / min; preferably, the solution obtained in step (1) is added dropwise into the calcium chloride solution at a rate of 10 mL / min.

[0018] In one embodiment of the present invention, the concentration of calcium chloride solution in step (2) is 0.05-0.2 mol / L, preferably 0.1 mol / L.

[0019] The present invention also provides a method for inactivating Aeromonas hydrophila, wherein the method comprises mixing the composite photocatalytic antibacterial agent with Aeromonas hydrophila and inactivating Aeromonas hydrophila under light source irradiation.

[0020] In one embodiment of the present invention, the light source includes visible light.

[0021] In one embodiment of the present invention, the light source includes a white fluorescent lamp.

[0022] In one embodiment of the present invention, the power of the light source is 5-45W.

[0023] In one embodiment of the present invention, the irradiation time of the light source is 10-60 minutes.

[0024] In one embodiment of the present invention, the amount of the composite photocatalytic antibacterial agent added is 0.2-4 mg / mL.

[0025] In one embodiment of the present invention, the method involves adding a composite photocatalytic antibacterial agent to Aeromonas hydrophila, culturing in the dark, and then culturing under light.

[0026] In one embodiment of the present invention, the light-protected incubation time is 1-3 hours, preferably 2 hours.

[0027] In one embodiment of the present invention, the light incubation time is not less than 10 min, preferably 10-60 min, or preferably 30 min.

[0028] The present invention also provides a product containing the composite photocatalytic antibacterial agent, or the composite photocatalytic antibacterial agent prepared by the method.

[0029] The present invention also provides the composite photocatalytic antibacterial agent, or the application of the product in the inactivation of Aeromonas hydrophila not for the purpose of disease treatment or diagnosis.

[0030] Beneficial effects:

[0031] (1) This invention prepares a composite photocatalytic antibacterial agent, Em / SA, by loading emodin onto sodium alginate. Compared to using emodin alone, the composite photocatalytic antibacterial agent Em / SA can further enhance the inactivation effect on Aeromonas hydrophila cells. For bacterial concentrations of 10... 7 A suspension of Aeromonas hydrophila at cfu / mL, when treated with the combined photocatalytic antibacterial agent Em / SA (active ingredient 0.5 mg emodin), showed a decrease in the survival rate of Aeromonas hydrophila from 99.33% when emodin was used alone to less than 21.65% under conditions of 32W visible light as the light source and irradiation time of 30 min.

[0032] (2) Simple method and environmentally friendly: The preparation method of the supported photocatalytic antibacterial agent Em / SA is simple and green. The composite photocatalytic antibacterial agent Em / SA used in the process of photo-inactivating Aeromonas hydrophila contains only the natural products of Chinese herbal medicine, emodin and sodium alginate. The reaction is carried out under light and will not cause secondary pollution to the environment.

[0033] (3) High efficiency and time saving: This invention only requires adding Em / SA into the Aeromonas hydrophila cell suspension and irradiating it with white light for 30 minutes, which can efficiently achieve photoinactivation of Aeromonas hydrophila, reducing the survival rate to less than 21.65%. Attached Figure Description

[0034] Figure 1 Figure: Experimental results of minimum inhibitory concentration of emodin against Aeromonas hydrophila.

[0035] Figure 2(a) and (b) show the inhibitory effect of 256 mg / L emodin on Aeromonas hydrophila under dark conditions, and (c) and (d) show the inhibitory effect of 256 mg / L emodin on Aeromonas hydrophila under light conditions.

[0036] Figure 3 The inhibitory effect of different concentrations of emodin on Aeromonas hydrophila.

[0037] Figure 4 The inhibitory effects of different light sources on Aeromonas hydrophila.

[0038] Figure 5 Infrared spectra of sodium alginate, emodin, and Em / SA.

[0039] Figure 6 The inhibitory effect of the composite photocatalytic antibacterial agent Em / SA on Aeromonas hydrophila. Detailed Implementation

[0040] The present invention will be further illustrated below through examples.

[0041] The materials used in the following examples were purchased from Shanghai Sinopharm, and the strain Aeromonas hydrophila was purchased from the American Cell Culture Collection Center, with the strain number ATCC35654.

[0042] Culture medium formulation:

[0043] NB culture medium formula: 1 g / L beef extract, 2 g / L yeast extract, 5 g / L tryptone, 5 g / L sodium chloride.

[0044] NB solid culture medium formula: 1 g / L beef extract, 2 g / L yeast extract, 5 g / L tryptone, 5 g / L sodium chloride, 15 g / L agar powder.

[0045] All culture vessels and culture media used in this invention are sterilized at 121°C for 15 minutes.

[0046] Preparation of Aeromonas hydrophila suspension:

[0047] Frozen Aeromonas hydrophila were removed from a -80°C freezer, streaked onto NB agar plates, and incubated at 28°C for 16 hours. Subsequently, a single colony from the NB agar plates was inoculated into 5 mL of NB agar and incubated overnight at 28°C with a shaker. The next day, the overnight Aeromonas hydrophila colony was inoculated into 50 mL of NB agar and incubated at 28°C with a shaker for 2 hours. The bacterial cells were collected and diluted to a concentration of 10⁻⁶. 7 A suspension of Aeromonas hydrophila was obtained by dispersing cfu / mL, and the resulting suspension was used for subsequent antibacterial experiments.

[0048] Example 1: Preparation of the composite photocatalytic antibacterial agent Em / SA

[0049] (1) Dissolve 1g of sodium alginate (SA) in 100mL of water, stir well, add 10mg of emodin (Em), and continue stirring for 8h.

[0050] (2) Using a 1mL syringe, the solution obtained in step (1) was added dropwise to a 0.1M CaCl2 solution and washed three times with water. After pre-cooling at -80℃, it was dried in a freeze dryer to obtain the supported photocatalytic antibacterial agent Em / SA.

[0051] Infrared spectroscopy was performed on sodium alginate, emodin, and the composite photocatalytic antibacterial agent Em / SA in this embodiment. Figure 5 As shown, the composite photocatalytic antibacterial agent Em / SA contains characteristic peaks of both sodium alginate and emodin, indicating that the method of the present invention successfully synthesized the composite photocatalytic antibacterial agent Em / SA.

[0052] Example 2: Inactivation effect of the composite photocatalytic antibacterial agent Em / SA on Aeromonas hydrophila under light irradiation.

[0053] (1) Preparation of composite photocatalytic antibacterial agent

[0054] The preparation method is the same as in Example 1, except that in step (1), the amount of sodium alginate added is 50 mg and the amount of emodin added is 0.5 mg.

[0055] (2) Inactivation effect of composite photocatalytic antibacterial agent on Aeromonas hydrophila under light irradiation

[0056] Take 50.5 mg of the composite photocatalytic antibacterial agent obtained in step (1) (0.5 mg of emodin active ingredient), add it to 50 mL of Aeromonas hydrophila cell suspension, and then react under 32 W visible light for 30 min. After the reaction, take 100 μL of the sample into a centrifuge tube and dilute it with 0.9% NaCl solution. 5 The 100 μL sample was diluted and spread onto NB solid medium, then incubated at 28°C for 24 h. The cell count was then calculated and analyzed, and the results showed that the survival rate of Aeromonas hydrophila was 28.41%.

[0057] Comparative Example 1: The inactivation effect of emodin alone on Aeromonas hydrophila under light.

[0058] Add 0.5 mg of emodin to 50 mL of Aeromonas hydrophila cell suspension, and then react under 32 W visible light for 30 min. After the reaction, take 100 μL of the sample into a centrifuge tube and dilute it with 0.9% NaCl solution. 5The diluted 100 μL sample was plated onto NB solid medium and incubated at 28°C for 24 h. Cell counts were then calculated and analyzed. The results showed that the survival rate of *Aeromonas hydrophila* was 99.33%, indicating that emodin alone, under the above conditions, was not effective in inactivating *Aeromonas hydrophila*. Comparative Example 2: Inactivation effect of sodium alginate alone on *Aeromonas hydrophila* under light.

[0059] Add 50 mg of sodium alginate to 50 mL of Aeromonas hydrophila cell suspension, and then react under 32 W visible light for 30 min. After the reaction, take 100 μL of the sample into a centrifuge tube and dilute it with 0.9% NaCl solution. 5 The 100 μL sample was diluted and spread onto NB solid medium, then incubated at 28°C for 24 h. The cell count was calculated and analyzed. The results showed that the survival rate of Aeromonas hydrophila was 99.72%, indicating that sodium alginate alone could not inactivate Aeromonas hydrophila under the above conditions.

[0060] Comparative Example 3: Inactivation effect of emodin combined with sodium alginate on Aeromonas hydrophila under light.

[0061] 50 mg of sodium alginate and 0.5 mg of emodin were added directly to 50 mL of Aeromonas hydrophila cell suspension, and then reacted under 32 W visible light for 30 min. After the reaction, 100 μL of the sample was transferred to a centrifuge tube and diluted with 0.9% NaCl solution. 5 The diluted 100 μL sample was spread on NB solid medium and then incubated at 28°C for 24 h. The cell count was calculated and analyzed. The results showed that the survival rate of Aeromonas hydrophila was 99.48%, indicating that the direct combination of emodin and sodium alginate under light could not inactivate Aeromonas hydrophila under the above conditions.

[0062] Comparative Example 4: Inactivation effect of the composite photocatalytic antibacterial agent Em / CNF on Aeromonas hydrophila under light irradiation

[0063] (1) Preparation of the composite photocatalytic antibacterial agent Em / CNF

[0064] The preparation method is the same as in Example 1, except that in step (1), sodium alginate is replaced by cellulose nanofibers (CNF), the amount added is 50 mg, and the amount added by emodin is 0.5 mg.

[0065] (2) Inactivation effect of composite photocatalytic antibacterial agent on Aeromonas hydrophila under light irradiation

[0066] Take 50.5 mg of the composite photocatalytic antibacterial agent obtained in step (1) (0.5 mg of emodin active ingredient), add it to 50 mL of Aeromonas hydrophila cell suspension, and then react under 32 W visible light for 30 min. After the reaction, take 100 μL of the sample into a centrifuge tube and dilute it with 0.9% NaCl solution. 5 The diluted 100 μL sample was spread on NB solid medium and then incubated at 28 °C for 24 h. The cell count was calculated and analyzed. The results showed that the survival rate of Aeromonas hydrophila was 98.86%, indicating that the composite antibacterial agent Em / CNF could not inactivate Aeromonas hydrophila under light conditions. This suggests that not all three-dimensional porous materials can significantly improve the antibacterial effect of emodin.

[0067] Example 3: Inactivation effect of the composite photocatalytic antibacterial agent Em / SA prepared with different amounts of emodin on Aeromonas hydrophila under light irradiation.

[0068] (1) Preparation of the composite photocatalytic antibacterial agent Em / SA-1

[0069] The preparation method is the same as in Example 1, except that the amount of sodium alginate added in step (1) is 1g and the amount of emodin added is 50mg. The resulting composite photocatalytic antibacterial agent is Em / SA-1.

[0070] (2) Preparation of composite photocatalytic antibacterial agent Em / SA with different amounts of emodin

[0071] Following the method in step (1) above, the difference is that the amount of emodin added is 100 mg. The composite photocatalytic antibacterial agent Em / SA-2 was prepared.

[0072] Following the method in step (1) above, the difference is that the amount of emodin added is 150 mg. The composite photocatalytic antibacterial agent Em / SA-3 was prepared.

[0073] Following the method in step (1) above, the difference is that the amount of emodin added is 200 mg. The composite photocatalytic antibacterial agent Em / SA-4 was prepared.

[0074] (3) Take 50.5 mg of each of the composite photocatalytic antibacterial agents Em / SA-1, Em / SA-2, Em / SA-3, and Em / SA-4 prepared in step (2), add them to 50 mL of Aeromonas hydrophila cell suspension, and then react under 32 W visible light for 30 min. After the reaction, take 100 μL of the sample into a centrifuge tube and dilute it with 0.9% NaCl solution. 5The diluted 100 μL sample was spread onto NB solid medium and incubated at 28 °C for 24 h. Cell counts were then calculated and analyzed. The results showed that the survival rates of Aeromonas hydrophila were 21.65%, 15.44%, 9.64%, and 3.27%, respectively. Figure 6 ).

[0075] Comparative Example 4: Minimum inhibitory concentration of emodin against Aeromonas hydrophila

[0076] (1) Prepare emodin solutions with concentrations of 2048, 1024, 512, 256, 128, 64, 32, 16 and 8 mg / L respectively;

[0077] (2) Take Aeromonas hydrophila from the preservation tube and inoculate it into 100 μL of NB medium (at which point the initial OD) 600 (close to 0);

[0078] (3) Add 100 μL of the different concentrations of emodin solution obtained in step (1) to the culture medium of step (2), and incubate at 28℃ and 150 rpm for 24 h. Measure the absorbance at 600 nm. Figure 1 As shown, when the amount of emodin added is less than 256 mg / L, the OD of Aeromonas hydrophila... 600 Compared to the initial OD 600 A significant increase was observed, indicating that when the amount of emodin added was less than 256 mg / L, it could not effectively inactivate Aeromonas hydrophila. When the amount of emodin added was 256 mg / L or higher, the OD of Aeromonas hydrophila increased significantly. 600 Compared to the initial OD 600 The lack of significant changes indicates that only when the amount of emodin added is 256 mg / L or higher can it effectively inactivate Aeromonas hydrophila. The minimum effective inhibitory concentration of emodin is 256 mg / L.

[0079] Comparative Example: Inactivation Effect of Five Emodins on Aeromonas hydrophila in the Dark

[0080] 12.8 mg of emodin was added to 50 mL of Aeromonas hydrophila cell suspension, and then incubated in the dark for 15, 30, and 60 min respectively. After the reaction, 100 μL of the sample was transferred to a centrifuge tube and diluted with 0.9% NaCl solution. 5 The diluted 100 μL sample was spread onto NB solid medium and incubated at 28°C for 24 h. Cell counts were then calculated and analyzed. The results showed that the survival rates of Aeromonas hydrophila were 97.89%, 98.63%, and 99.46%, respectively. Figure 2 This indicates that emodin cannot inactivate Aeromonas hydrophila in the dark.

[0081] Comparative Example: Inactivation Effect of 6 Emodins on Aeromonas hydrophila under Different Light Exposure Times

[0082] 12.8 mg of emodin was added to 50 mL of Aeromonas hydrophila cell suspension, and then incubated under 32 W visible light for 15, 30, and 60 min respectively. After the reaction, 100 μL of the sample was transferred to a centrifuge tube and diluted with 0.9% NaCl solution. 5 The diluted 100 μL sample was spread onto NB solid medium and incubated at 28 °C for 24 h. Cell counts were then calculated and analyzed. The results showed that the survival rates of Aeromonas hydrophila were 67.89%, 13.76%, and 10.38%, respectively. Figure 2 ).

[0083] Comparative Example 7: Inactivation effect of different amounts of emodin on Aeromonas hydrophila under light.

[0084] 6.4 mg, 3.2 mg, 1.6 mg, and 0.8 mg of emodin were added to 50 mL of Aeromonas hydrophila cell suspension, respectively, and reacted under 32 W visible light for 30 min. After the reaction, 100 μL of the sample was transferred to a centrifuge tube and diluted with 0.9% NaCl solution. 5 The diluted 100 μL sample was spread onto NB solid medium and incubated at 28 °C for 24 h. Cell counts were then calculated and analyzed. The results showed that the survival rates of Aeromonas hydrophila were 16.72%, 25.4%, 51.86%, and 90.35%, respectively. Figure 3 ).

[0085] Comparative Example: Inactivation Effect of 8 Emodins on Aeromonas hydrophila under Different Light Power Irradiations

[0086] 6.4 mg of emodin was added to 50 mL of Aeromonas hydrophila cell suspension, and then reacted under visible light at 5 W, 15 W, 23 W, 32 W, and 45 W for 30 min, respectively. After the reaction, 100 μL of the sample was transferred to a centrifuge tube and diluted with 0.9% NaCl solution. 5 The diluted 100 μL sample was spread onto NB solid medium and incubated at 28 °C for 24 h. Cell counts were then calculated and analyzed. The results showed that the survival rates of Aeromonas hydrophila were 66.34%, 42.35%, 31.86%, 16.72%, and 11.32%, respectively. Figure 4 ).

Claims

1. A composite photocatalytic antibacterial agent, characterized in that, The composite photocatalytic antibacterial agent is used to photocatalytically inactivate Aeromonas hydrophila, and the composite photocatalytic antibacterial agent is composed of sodium alginate and emodin as an active ingredient. The preparation method of the composite photocatalytic antibacterial agent includes the following steps: (1) Mix emodin with sodium alginate aqueous solution and stir; (2) Mix the solution obtained in step (1) with the calcium chloride solution; (3) Rinse the mixture obtained in step (2), pre-cool it, freeze-dry it, and obtain a composite photocatalytic antibacterial agent; In step (1), the mass ratio of emodin to sodium alginate is 1:(5-100).

2. The preparation method of the composite photocatalytic antibacterial agent according to claim 1, characterized in that, The method includes the following steps: (1) Mix emodin with sodium alginate aqueous solution and stir; (2) Mix the solution obtained in step (1) with the calcium chloride solution; (3) Rinse the mixture obtained in step (2), pre-cool it, freeze dry it to obtain a composite photocatalytic antibacterial agent.

3. The preparation method according to claim 2, characterized in that, In step (1), the sodium alginate aqueous solution is obtained by mixing sodium alginate with water and stirring to dissolve it, and the concentration of the sodium alginate aqueous solution is 5-20 g / L.

4. A method for inactivating Aeromonas hydrophila, characterized in that, The method involves mixing the composite photocatalytic antibacterial agent of claim 1, or the composite photocatalytic antibacterial agent prepared by the method of claim 2 or 3, with Aeromonas hydrophila, and then inactivating Aeromonas hydrophila under light source irradiation; the method is not intended for disease treatment or diagnosis.

5. The method according to claim 4, characterized in that, The amount of the composite photocatalytic antibacterial agent added is 0.2-4 mg / mL.

6. The method according to claim 5, characterized in that, The illumination time of the light source is not less than 10 minutes; the light source includes visible light.

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