CN115960379A

CN115960379A - Preparation method and application of antibacterial polylysine film

Info

Publication number: CN115960379A
Application number: CN202111179134.0A
Authority: CN
Inventors: 王亮; 王蕊; 刘晶
Original assignee: Tianjin University of Technology
Current assignee: Tianjin University of Technology
Priority date: 2021-10-11
Filing date: 2021-10-11
Publication date: 2023-04-14

Abstract

The invention discloses a preparation method and application of an antibacterial polylysine film. A series of reactions are carried out to successfully prepare a novel antibacterial polylysine film. Coating a lactic acid malic acid polymer on the surface of a polylactic acid-glycolic acid Polymer (PLGA) film to obtain a polylactic acid film, and performing a crosslinking reaction on the prepared polylactic acid film and polylysine hydrochloride (PLL) to prepare the antibacterial polylysine film. The invention proves the excellent antibacterial performance of the antibacterial polylysine film through an antibacterial experiment, and the invention has important significance for the development of novel antibacterial materials.

Description

Preparation method and application of antibacterial polylysine film

Technical Field

The invention relates to an antibacterial polylysine film, and belongs to the field of antibacterial films. Because polylactic acid has poor antibacterial performance and can grow mould after long-term use, the polylactic acid film and polylysine hydrochloride are combined by an ionic crosslinking method, and the antibacterial polylysine film is prepared by a simple preparation method.

Background

The antibacterial coating is widely applied to the fields of toys for children, furniture and the like, so that people are prevented from being infected. The natural antibacterial agents such as chitosan, mugwort, aloe and the like belong to natural extracts. In life, most of the antibiotics are used, and the aim of sterilization is achieved by methods of blocking protein synthesis required by bacterial growth, increasing membrane permeability, losing nutrients in thalli and the like. However, the large blind use of antibiotics has caused many bacteria to generate drug resistance, which is not a good development trend, and although natural biological antibacterial agents have better compatibility to human beings, lower toxicity and weaker damage to the environment, the antibacterial ability is relatively poor, and the types of bacteria capable of being killed are not many. Therefore, there is a need to develop new antibacterial materials to meet the needs of human production and living.

Polylactic-co-glycolic acid (PLGA) is a degradable functional polymer organic compound, has good biocompatibility, no toxicity and good properties of encapsulation and film formation, and is widely applied to the fields of pharmacy, medical engineering materials and modern industry. The degradation products of PLGA are lactic acid and glycolic acid, which are also by-products of the human metabolic pathway, and thus it has no side effects when applied in medicine and biomaterials.

The study of polylysine, a natural product of biological metabolism, has been relatively well established abroad, particularly in Japan. Not only has good sterilization capability and thermal stability, but also has excellent antiseptic property and huge commercial potential. In japan, polylysine has been approved as a preservative for addition to foods and is widely used in the preservation of instant rice, wet-boiled noodles, boiled dishes, seafood, sauces, soy sauce, fish fillet, and cookies. Meanwhile, the polylysine is matched with other natural bacteriostatic agents for use, so that the remarkable synergistic effect is achieved, and the bacteriostatic ability of the bacteriostatic agent can be improved. The polylysine mechanism is mainly characterized by the destruction of the cell membrane structure of microorganisms, causing the interruption of the mass, energy and information transmission of cells, eventually leading to cell death.

Here, we provide a polylactic acid film and combine it with polylysine hydrochloride by means of ionic crosslinking to prepare a highly effective antibacterial polylysine film.

Disclosure of Invention

The inventive concept consists of two parts. The first is polymer PLMA for synthesizing lactic acid malic acid; and secondly, the polylactic acid film and the polylysine hydrochloride are subjected to ion crosslinking to prepare the antibacterial polylysine film.

The invention aims to provide a synthetic method of a novel antibacterial polylysine film. The antibacterial film has higher antibacterial activity, and opens up a new way for developing a new high-efficiency antibacterial coating.

The technical scheme of the invention is as follows:

the preparation method of the PLMA comprises the following steps:

1) Preparation of PLMA: adding lactic acid into a reaction bottle, distilling under reduced pressure to obtain light yellow product, adding malic acid into the low-molecular lactic acid, and continuously stirring for reacting for 24h.

2) Post-treatment of the PLMA: washing the reacted product with diethyl ether and tetrahydrofuran for three times, pouring out the upper layer solvent, and drying the lower layer white viscous product through a vacuum drying oven to obtain the PLMA.

The preparation method of the antibacterial polylysine film comprises the following steps:

1) Preparation of polylactic acid film: firstly, completely dissolving PLGA in chloroform, preparing a PLGA film by adopting a tape casting method, then dissolving PLMA in acetone, coating the solution on the surface of the prepared PLGA film, and drying in vacuum to obtain the polylactic acid film.

2) Preparation of an antibacterial polylysine film: firstly, soaking the prepared polylactic acid film in a sodium hydroxide solution, then taking out the polylactic acid film, soaking the polylactic acid film in a polylysine hydrochloride aqueous solution for a period of time, taking out the film, naturally airing the film at room temperature, and then drying the film in a vacuum drying oven to obtain the antibacterial polylysine film.

The obtained antibacterial polylysine film can be used for antibacterial coating.

Technical analysis of the invention:

PLMA is synthesized by the copolycondensation of lactic acid and malic acid, which includes both intermolecular and intramolecular dehydration. And the polylysine hydrochloride serving as a polycation antibacterial agent is crosslinked with the polylactic acid film to obtain the antibacterial polylactic acid film. After the polylysine acts on cells, the cell structure is gradually destroyed, and finally the cells die, thereby playing a role in bacteriostasis.

In conclusion, the invention provides a preparation method of a novel antibacterial polylysine film, and the polylysine antibacterial film is a promising antibacterial material. The coating has good chemical stability and strong antibacterial property, and has good antibacterial property on Escherichia coli and Staphylococcus aureus.

The invention has the advantages and beneficial effects that:

1) The reaction mechanism involved in the synthesis of the PLMA part is simple and mature, namely, the copolycondensation reaction of the lactic acid and the malic acid is realized, and the treatment process after the reaction is simpler and easier to operate.

2) The prepared antibacterial polylysine film has high antibacterial activity and good antibacterial performance on escherichia coli and staphylococcus aureus.

3) The preparation method of the prepared antibacterial polylysine film is simple and easy to implement.

Drawings

Figure 1 is a scheme of the synthesis scheme for lactic acid malic acid Polymer (PLMA).

FIG. 2 is a diagram of a method for preparing an antibacterial polylysine film.

Fig. 3 is a water contact angle of the antibacterial polylysine film.

FIG. 4 is a comparison of the antibacterial polylysine film against E.coli with different bacterial liquid concentrations.

FIG. 5 is a graph comparing the use of antibacterial polylysine films against different concentrations of Staphylococcus aureus.

FIG. 6 is a drawing of an antimicrobial polylysine film pair 10 ⁸ Time comparison plot of CFU/mL E.coli.

FIG. 7 shows a pair of antimicrobial polylysine films 10 ⁸ Time comparison plot of CFU/mL S.aureus.

Detailed Description

Example 1

Preparation of lactic acid malic acid Polymer (PLMA):

preparation method of lactic acid malic acid Polymer (PLMA):

10mL of lactic acid is added into a 100mL round-bottom bottle, heated and refluxed in an oil bath, and reacted for 12 hours at 150 ℃ to obtain a light yellow viscous product, namely, oligolactic acid (PLA). Then 5g malic acid was added and reacted at 150 ℃ for 24h. After the reaction is finished, cooling the reaction to room temperature, dissolving the obtained product by tetrahydrofuran, adding anhydrous ether, washing the product by the anhydrous ether and the tetrahydrofuran for three times, pouring out the upper-layer solvent, and then putting the lower-layer white viscous product into a vacuum drying oven at 45 ℃ for drying for 48 hours to obtain the product PLMA.

The synthetic route of lactic acid malic acid Polymer (PLMA) is shown in figure 1.

Example 2

Preparation of polylysine film:

the preparation method of the polylysine film comprises the following steps:

1) Dissolving PLMA in acetone to prepare 4% solution, spreading on the surface of the PLGA film by a tape casting method, naturally drying at room temperature for 24h after film formation, and then drying in a vacuum drying oven at 40 ℃ for 48h to obtain the polylactic acid film.

2) Soaking the prepared polylactic acid film in 0.1mol/L sodium hydroxide solution for 1min, taking out the polylactic acid film, soaking the polylactic acid film in 0.2mol/L polylysine hydrochloride aqueous solution for 30min, taking out the film after reaction, naturally drying the film at room temperature, and then drying the film in a vacuum drying oven at 40 ℃ for 48h to obtain the antibacterial polylysine film.

The preparation process of the antibacterial polylysine film is shown in figure 2, and the water contact angle of the antibacterial polylysine film is shown in figure 3.

Example 3

Antibacterial activity test of antibacterial polylysine films:

the prepared antibacterial polylysine film is dripped with bacterial liquid in the central area, the relation between the antibacterial performance of the film and the time and the concentration is researched, and the antibacterial performance to escherichia coli (E.coli) and staphylococcus aureus (S.aureus) is researched. The antibacterial activity of the antibacterial agent is counted by a colony counting method, and then the bacteriostasis rate is calculated. The dripping concentration in the central area is respectively 10 ⁹ CFU/mL、10 ⁸ CFU/mL、10 ⁷ CFU/mL、10 ⁶ CFU/mL and 10 ⁵ Culturing CFU/mL E.coli or S.aureus bacterial solution in 37 deg.C incubator for 30min, ultrasonically treating in 2mL sterile physiological saline for 2min, uniformly coating the bacteria solution on solid culture medium, culturing in 37 deg.C incubator, and concentrating different bacterial solutionsComparative plots of degrees of E.coli and S.aureus (FIG. 4, FIG. 5). Selecting the bacteria solution with the concentration of 10 according to the results ⁸ CFU/mL e.coli or s.aureus as the subject of subsequent antibacterial and time-action relationships.

Dripping the antibacterial polylysine film to the central area of the antibacterial polylysine film at a concentration of 10 ⁸ CFU/mL of e.coli or s.aureus bacterial solution, cultured in a 37 ℃ incubator for 5min, 10min, 15min, 20min, 25min, respectively, then coated on a solid medium, cultured in a 37 ℃ incubator, and the results obtained by the colony counting method are shown in fig. 6 and fig. 7.

The antibacterial result shows that:

1) Coli and s.aureus for different concentrations in fig. 4 and 5, it can be seen that the pair of antibacterial polylysine films 10 at an action time of 30min ⁵ CFU/mL、10 ⁶ CFU/mL、10 ⁷ CFU/mL、10 ⁸ The antibacterial activity of E.coli and S.aureus of CFU/mL reaches more than 99.9 percent, but the antibacterial activity of the E.coli and S.aureus of CFU/mL reaches 10 percent ⁹ The antibacterial property of the bacterial liquid of CFU/mL is only 68.4%.

2) For staphylococcus aureus: in the bacterial liquid concentration of 10 ⁸ When the action time is 5min, 10min, 15min, 20min and 25min respectively at CFU/mL, the result shows that the bacteriostasis rate to staphylococcus aureus is 56.4% at 5min and 68.7% at 10min (as shown in figure 7). Along with the increase of time, the antibacterial activity of the antibacterial polylysine film to staphylococcus aureus is gradually enhanced, and the antibacterial activity reaches more than 99.9 percent when the action time is 20min (as shown in figure 7).

3) For E.coli: the concentration of the Escherichia coli liquid is 10 ⁸ At CFU/mL, the action time in a constant temperature incubator at 37 ℃ is respectively 5min, 10min, 15min, 20min and 25min, and the result shows that the bacteriostasis rate is only 25.8% at 5min, the antibacterial activity of the antibacterial polylysine film on escherichia coli is positively correlated with the time, and the antibacterial activity reaches more than 99.9% at the action time of 20min (as shown in figure 6).

4) According to the test of the antibacterial performance of the antibacterial polylysine film pair to e.coli and s.aureus (fig. 6, fig. 7), the results show that the antibacterial polylysine film pair 10 ⁸ E.co of CFU/mLThe antibacterial activity of li is stronger than that of S.aureus, and the antibacterial activity of both reaches 100% in 25 min.

Claims

1. A preparation method of an antibacterial polylysine film is characterized by comprising the following steps:

1) Step 1: the preparation process of the lactic acid malic acid Polymer (PLMA) comprises the steps of heating lactic acid in an oil bath for reflux reaction to obtain a light yellow product, namely oligolactic acid, adding malic acid for continuous reaction to obtain the lactic acid malic acid Polymer (PLMA);

2) Dissolving the PLMA obtained in the step 1) and coating the dissolved PLMA on the surface of a polylactic-co-glycolic acid (PLGA) film to obtain a polylactic acid film;

3) And (3) carrying out a cross-linking reaction on the polylactic acid film obtained in the step 2) and polylysine hydrochloride (PLL) to obtain the antibacterial polylysine film.

2. The method of preparing an antibacterial polylysine film according to claim 1, wherein: the step 2) comprises the following steps: the solvent used for the PLMA dissolution was acetone.

3. The method for preparing an antibacterial polylysine film according to claim 1, wherein: the step 3) also comprises the step of forming the antibacterial polylysine film by taking the polylactic acid film as an anion cross-linking agent and taking PLL as polycation through ionic crosslinking.

4. The antibacterial polylysine film according to claim 3, which is used for sterilization.