A modified polyvinyl alcohol and chitosan composite film material for killing bacteria and adsorbing antibiotic resistance genes, and a preparation method

Abstract

The application discloses a modified polyvinyl alcohol and chitosan composite film material for sterilization and adsorption of antibiotic resistance genes, and a preparation method thereof, and belongs to the technical field of sewage treatment materials.The modified polyvinyl alcohol and chitosan composite film material is prepared from polyvinyl alcohol (PVA), chitosan (CS) and long-chain quaternary ammonium salt modified polyvinyl alcohol as raw materials, mixed, evaporated and dried, and then the film is obtained.The composite film material is mixed with a bacterial solution at room temperature, the long-chain quaternary ammonium salt can destroy the cell membrane and the adsorption of the film to the antibiotic resistance genes in the solution, and therefore, the antibiotic resistance genes in and outside the cells can be effectively removed synchronously, and the method has the advantages of simple technology and no secondary pollution.

Description

Technical Field

[0001] This invention belongs to the field of wastewater treatment materials technology, specifically relating to a modified polyvinyl alcohol and chitosan composite membrane material, its preparation method, and its application. Background Technology

[0002] Antibiotic resistance genes (ARGs) play a crucial role in the formation of superbugs, thereby affecting the clinical efficacy of antibiotic treatment and posing a significant threat and challenge to public health. In recent years, antibiotic resistance genes have been considered an emerging pollutant. Unlike conventional pollutants, antibiotic resistance genes are DNA segments encoding antibiotic resistance proteins, existing in both intracellular and extracellular forms. The two types of antibiotic resistance genes spread differently: extracellular antibiotic resistance genes can remain stable in different environmental media for up to several months, entering naturally occurring competent cells through transformation; intracellular antibiotic resistance genes can amplify as the host microorganism multiplies and can also spread between different microbial hosts through horizontal gene transfer mediated by mobile gene units.

[0003] However, traditional wastewater treatment technologies are designed for nutrients such as nitrogen (N) and phosphorus (P) in water bodies, and their effectiveness in removing antibiotic resistance genes is minimal. Traditional water treatment methods, including chlorination, ozone, and ultraviolet disinfection, cannot effectively destroy antibiotic resistance genes and may even release them into the environment by damaging bacterial cell membranes. Furthermore, although 90%–99% of microorganisms are effectively removed from treated urban wastewater, the absolute number of bacteria remains high, with total coliform counts between 10⁴ and 10⁵ CFU / L, and pathogenic and drug-resistant bacteria are also present. Studies have shown that the discharge of reclaimed water into environmental water bodies leads to an increase in the abundance of antibiotic resistance genes in the receiving water or soil environment. During the reuse of reclaimed water, it is necessary to monitor the presence of antibiotic resistance genes to prevent reclaimed water from becoming a medium for the spread of antibiotic resistance genes.

[0004] Antibiotic resistance genes and competent bacteria carrying these genes are prevalent in water bodies such as medical and domestic wastewater, polluting the environment and endangering human health. Most existing methods aim to remove pathogens by disrupting their cell membranes. However, the antibiotic resistance genes released from these damaged membranes are released into the water, continuing to harm human health. Therefore, simultaneous sterilization and removal of antibiotic resistance genes is crucial. Patent applications CN202311417225 and CN202311488893 report methods for sterilization and removal of antibiotic resistance genes. How to efficiently and environmentally eliminate and remove antibiotic resistance genes is of significant practical importance and demand. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention aims to design and provide a method for preparing and applying a modified polyvinyl alcohol (PVA) and chitosan composite membrane material. The method of the present invention uses a long-chain quaternary ammonium salt-modified PVA solution as a base, and chitosan and PVA solutions as carriers to prepare a long-chain quaternary ammonium salt-modified PVA / chitosan composite membrane material. This material can sterilize pathogenic bacteria at room temperature, causing the bacterial cell membranes to rupture and die, and adsorbing antibiotic resistance genes released into the water by the dead pathogens, achieving simultaneous sterilization and adsorption / removal of antibiotic resistance genes in a short time.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] On one hand, the present invention provides a modified polyvinyl alcohol and chitosan composite membrane material, comprising a polyvinyl alcohol and chitosan composite membrane and a long-chain quaternary ammonium salt modified polyvinyl alcohol membrane.

[0008] Secondly, the present invention provides a method for preparing a modified polyvinyl alcohol and chitosan composite film material, comprising the following steps:

[0009] (1) Weigh chitosan solution and polyvinyl alcohol solution, control the temperature and stir, add crosslinking agent (to enhance the film-forming properties of the membrane solution), heat and stir again to obtain polyvinyl alcohol and chitosan composite membrane solution; use chitosan and polyvinyl alcohol solution as film-forming agents;

[0010] (2) Weigh the polyvinyl alcohol solution, add epichlorohydrin, treat at high temperature, add long-chain quaternary ammonium salt, heat and stir to achieve quaternary ammonium salt loading, and obtain long-chain quaternary ammonium salt modified polyvinyl alcohol film solution.

[0011] (3) Weigh the polyvinyl alcohol and chitosan composite membrane liquid and the long-chain quaternary ammonium salt modified polyvinyl alcohol membrane liquid, stir and mix them, and then sonicate them to make the membrane liquid evenly mixed. Place them in a petri dish, dry them and peel off the membrane to obtain the modified polyvinyl alcohol and chitosan composite membrane material.

[0012] In the preparation method described above, the temperature control in step (1) is 40-45℃;

[0013] The stirring time is 4 to 6 hours;

[0014] The heating and stirring conditions are: temperature 40-45℃, stirring time 4-6h;

[0015] The crosslinking agent is glutaraldehyde and glycerol;

[0016] The volume-to-mass ratio of the chitosan solution, polyvinyl alcohol solution, and crosslinking agent is 100 mL: 75 mL: 3 g;

[0017] Preferably, the crosslinking agent is glutaraldehyde and glycerol, with a mass ratio of glutaraldehyde to glycerol of 1:5.

[0018] The preparation method described above, the preparation method of the chitosan solution in step (1) is as follows: weigh chitosan, stir at room temperature, dissolve in acetic acid solution, and prepare chitosan solution;

[0019] The mass-to-volume ratio of chitosan to acetic acid solution is 5-8 g: 125-200 mL;

[0020] The concentration of the acetic acid solution is 2-5%.

[0021] The preparation method described above, in step (1), is as follows: weigh polyvinyl alcohol, heat and stir at 90-95°C to dissolve in water, and prepare a 40 g / L polyvinyl alcohol solution.

[0022] The preparation method described above, in step (2), the conditions for high-temperature treatment are: temperature 80-85℃, time 2-3h;

[0023] The heating and stirring conditions are: temperature 80-85℃, time 3-4h.

[0024] In the preparation method described above, the long-chain quaternary ammonium salt in step (2) is selected from one of NN-dimethyldodecaneamine, NN-dimethyltetradecaneamine, or NN-dimethylhexadecaneamine;

[0025] The volume ratio of the polyvinyl alcohol solution, epichlorohydrin, and long-chain quaternary ammonium salt is 3:1 to 2:1 to 2.

[0026] In the preparation method described above, the stirring and mixing time in step (3) is 2 to 5 minutes;

[0027] The ultrasound conditions are: duration 30–60 min, repeated 3–5 times.

[0028] In the preparation method described above, the mass ratio of the polyvinyl alcohol and chitosan composite film liquid and the long-chain quaternary ammonium salt modified polyvinyl alcohol film liquid in step (3) is 1:7 to 10.

[0029] The drying conditions are: temperature 30-40℃, time 72-96h.

[0030] Thirdly, the present invention provides the application of the modified polyvinyl alcohol and chitosan composite membrane material, or the modified polyvinyl alcohol and chitosan composite membrane material prepared by the method, in the treatment of wastewater for sterilization and adsorption of antibiotic resistance genes.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The modified chitosan / polyvinyl alcohol composite membrane material of the present invention can simultaneously sterilize and adsorb antibiotic resistance genes in wastewater when treating wastewater.

[0033] 2. This invention uses chitosan and polyvinyl alcohol as film-forming agents and quaternary ammonium salts as modifiers to prepare long-chain quaternary ammonium salt-modified chitosan / polyvinyl alcohol composite membrane materials under the action of epichlorohydrin. This offers structural advantages in wastewater treatment.

[0034] 3. The modified chitosan and polyvinyl alcohol composite membrane material of this invention can be applied to wastewater treatment for sterilization and adsorption of antibiotic resistance genes. At 37°C, the composite membrane material is mixed with bacterial solution, and the long-chain quaternary ammonium salt disrupts the cell membrane and the membrane's adsorption of antibiotic resistance genes in the solution. This composite material can be used at room temperature without additional heat; it can be achieved through shaking and stirring. The reaction cycle is 24 hours, with a sterilization rate exceeding 99%. Furthermore, the composite membrane material exhibits low loss rate, low energy consumption, high efficiency, and no secondary pollution, effectively achieving simultaneous sterilization and removal of intracellular and extracellular antibiotic resistance genes. Attached Figure Description

[0035] Figure 1 Here is a scanning electron microscope (SEM) image of the 12C modified membrane material obtained in Example 1;

[0036] Figure 2 The sterilization rates of the three membrane materials;

[0037] Figure 3 The adsorption rates of antibiotic resistance genes for the three membrane materials are shown. Detailed Implementation

[0038] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0039] Example 1:

[0040] A method for preparing a modified polyvinyl alcohol and chitosan composite film material includes:

[0041] (1) Weigh 4g of chitosan and dissolve it in 100mL of 2% acetic acid solution; then weigh 40g of polyvinyl alcohol solution, heat and stir at 95℃ to dissolve in water, and make up to 1L to prepare a 40g / L polyvinyl alcohol solution; take 75mL of polyvinyl alcohol solution and add it to 100mL of chitosan solution, heat and stir at 40℃ for 4h, then add 2g of glycerol and 0.5g of 50% glutaraldehyde solution, and continue to heat and stir for 4h to obtain a composite film solution of polyvinyl alcohol and chitosan, denoted as PVA / CS.

[0042] (2) Take 75 mL of 40 g / L polyvinyl alcohol solution, add 12.5 mL of epichlorohydrin, heat at 85 °C for 2 h, then add 12.5 mL of N-N-dimethyldodecaneamine, heat for 3 h to obtain a long-chain quaternary ammonium salt modified polyvinyl alcohol solution, denoted as 12C-PVA.

[0043] (3) Take 2 mL of PVA / CS solution and 14 mL of 12C-PVA, vortex for 2 min, sonicate for 30 min, repeat 3 times to ensure thorough mixing. Take 20 mL of the fully mixed solution and place it in a 90 mm petri dish, cover with a film, heat in an oven at 40℃ for 72 h, then peel off the film and wipe off any surface residue to obtain the modified polyvinyl alcohol and chitosan composite membrane material. The scanning electron microscope image of the composite membrane material is shown below. Figure 1 As shown.

[0044] Example 2:

[0045] The other steps are the same as in Example 1, except that in step (2), "add 12.5 mL of N-N-dimethyldodecaneamine" is changed to "add 12.5 mL of N-N-dimethyltetradecaneamine".

[0046] Example 3:

[0047] The other steps are the same as in Example 1, except that in step (2), "add 12.5 mL of N-N-dimethyldodecaneamine" is changed to "add 12.5 mL of N-N-dimethylhexadecaneamine".

[0048] Example 4:

[0049] Take 0.1g, 0.3g, and 0.5g of the modified polyvinyl alcohol and chitosan composite membrane materials from Examples 1, 2, and 3, respectively, and add them to 100mL of OD. 600 In a *E. coli* bacterial suspension with a concentration of 1, the control group consisted of *E. coli* bacterial suspension without the added modified composite membrane material. After reacting at 37°C and 180 rpm for 24 hours, 100 μL of the bacterial suspension was taken and plated to observe the sterilization effect. The results are as follows: Figure 2 As shown, the sterilization rates of the three modified polyvinyl alcohol and chitosan composite film materials in Examples 1, 2, and 3 are all above 90%. Specifically, the sterilization rate of the long-chain quaternary ammonium salt modified polyvinyl alcohol / chitosan composite film material obtained in Example 1 reached 100% at an addition amount of 0.1g; the sterilization rate of the long-chain quaternary ammonium salt modified polyvinyl alcohol / chitosan composite film material obtained in Example 2 reached 100% at an addition amount of 0.3g; and the sterilization rate of the long-chain quaternary ammonium salt modified polyvinyl alcohol / chitosan composite film material obtained in Example 3 reached 99% at an addition amount of 0.5g.

[0050] Example 5:

[0051] Take 0.1g, 0.3g, and 0.5g of the modified polyvinyl alcohol and chitosan composite membrane materials from Examples 1, 2, and 3, respectively, and add them to 100mL of OD. 600 In a culture of competent *E. coli* carrying an antibiotic resistance gene plasmid with a concentration of 1, and a control group of *E. coli* without modified composite membrane material, the culture was reacted at 37°C and 180 rpm for 24 hours. The culture was then centrifuged, and the supernatant was used for qPCR to detect the gene concentration. 100 μL of the culture was then plated to observe the sterilization effect. The results are as follows: Figure 3 As shown, the adsorption rates of the three membrane materials for antibiotic resistance genes all reached over 70%. Here, 12C refers to the composite membrane material prepared in Example 1, 14C refers to the composite membrane material prepared in Example 2, and 16C refers to the composite membrane material prepared in Example 3.

[0052] The qPCR testing method is as follows: The sample preparation method for qPCR detection is as follows: The entire qPCR sample system is 20 μL, and a 96-well PCR plate is required. Each system includes: 2 μL of sample DNA, 0.5 μL of upper primer (tetM-F, nucleotide sequence as shown in SEQ ID NO.1), 0.5 μL of lower primer (tetM-R, nucleotide sequence as shown in SEQ ID NO.2), 7 μL of ddH2O, and 10 μL of SYBR Green PCR premix. After adding all samples, mix well, centrifuge, and store on ice until testing. Each sample needs to be replicated in triplicate, and a negative control is required. The qPCR instrument test program is set as follows: preheating 95℃ for 150 s, denaturation 94℃ for 15 s, extension 56℃ for 30 s, for a total of 40 cycles. The nucleotide sequences of the primers are shown in Table 1 below.

[0053] Table 1. Basic information on primers used in the experiment.

[0054]

Claims

1. A method for preparing a modified polyvinyl alcohol and chitosan composite film material, characterized in that, The composite membrane material is formed by using chitosan and polyvinyl alcohol as carriers, and then composited with long-chain aliphatic tertiary amine-modified polyvinyl alcohol on the carriers; the long-chain aliphatic tertiary amine is selected from one of NN-dimethyldodecaneamine, NN-dimethyltetradecaneamine or NN-dimethylhexadecaneamine; The preparation method of the modified polyvinyl alcohol and chitosan composite film material includes the following steps: (1) Weigh out the acetic acid solution of chitosan and the aqueous solution of polyvinyl alcohol, control the temperature at 40~45℃ and stir for 4~6h, add glutaraldehyde and glycerol as crosslinking agents, and heat and stir at 40~45℃ for 4~6h to obtain the polyvinyl alcohol and chitosan composite film solution; the volume and mass ratio of the chitosan solution, polyvinyl alcohol solution and crosslinking agent is 100mL:75mL:3g; the mass ratio of glutaraldehyde and glycerol is 1:5; (2) Weigh an aqueous solution of polyvinyl alcohol, add epichlorohydrin, heat and react at 80-85°C for 2-3 hours, then add a long-chain aliphatic tertiary amine, heat and stir at 80-85°C for 3-4 hours to obtain a polyvinyl alcohol film solution modified with a long-chain aliphatic tertiary amine; the volume ratio of the polyvinyl alcohol solution, epichlorohydrin and long-chain aliphatic tertiary amine is 3:1-2:1-2; (3) Weigh the polyvinyl alcohol and chitosan composite film liquid and the long-chain fatty tertiary amine modified polyvinyl alcohol film liquid, shake and mix them, sonicate them, place them in a petri dish, dry them and peel off the film to obtain the modified polyvinyl alcohol and chitosan composite film material; the mass ratio of the polyvinyl alcohol and chitosan composite film liquid and the long-chain fatty tertiary amine modified polyvinyl alcohol film liquid is 1:7~10.

2. The preparation method according to claim 1, characterized in that, The method for preparing the chitosan solution in step (1) is as follows: weigh chitosan, stir at room temperature, dissolve in acetic acid solution, and prepare chitosan solution; The mass-to-volume ratio of chitosan to acetic acid solution is 5-8g:125-200mL; The concentration of the acetic acid solution is 2-5%.

3. The preparation method according to claim 1, characterized in that, The method for preparing the polyvinyl alcohol solution in step (1) is as follows: weigh polyvinyl alcohol, heat and stir at 90~95℃ to dissolve in water, and prepare a 40g / L polyvinyl alcohol solution.

4. The preparation method according to claim 1, characterized in that, The mixing time in step (3) is 2-5 minutes; The ultrasound conditions are: duration 30-60 minutes, repeated 3-5 times.

5. The preparation method according to claim 1, characterized in that, The drying conditions described in step (3) are: temperature 30~40℃, time 72~96h.

6. The application of the modified polyvinyl alcohol and chitosan composite membrane material prepared by the method according to any one of claims 1-5 in the sterilization and adsorption of antibiotic resistance genes in wastewater treatment.

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