CN112679925B

CN112679925B - Composite antibacterial film and preparation method and application thereof

Info

Publication number: CN112679925B
Application number: CN202011525224.6A
Authority: CN
Inventors: 曹立冬; 陈歌; 黄啟良; 曹冲; 赵鹏跃; 郑永权
Original assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Priority date: 2020-12-22
Filing date: 2020-12-22
Publication date: 2021-11-23
Anticipated expiration: 2040-12-22
Also published as: CN112679925A

Abstract

The invention provides a composite antibacterial film and a preparation method and application thereof, belonging to the technical field of film materials and being formed by casting a mixed solution, wherein the mixed solution comprises poly (3-hydroxybutyrate-co-4-hydroxybutyrate), prothioconazole and an organic solvent; the mass ratio of the poly (3-hydroxybutyrate-co-4-hydroxybutyrate) to the prothioconazole is (1.0-2.0): (0.01-1.0). According to the invention, the mass ratio of the poly (3-hydroxybutyrate-co-4-hydroxybutyrate) and the prothioconazole in the mixed solution is reasonably controlled, so that the doping amount of the prothioconazole in the film can ensure the antibacterial property of the film, does not influence the growth and development of plants, can be used for plant potting, and the application range of the prothioconazole is expanded.

Description

Composite antibacterial film and preparation method and application thereof

Technical Field

The invention relates to the technical field of film materials, in particular to a composite antibacterial film and a preparation method and application thereof.

Background

Poly (3-hydroxybutyrate-co-4-hydroxybutyrate) (P (3HB-co-4HB), hereinafter referred to as PHB) is a type of aliphatic polyester accumulated in prokaryotic microorganisms. As a biodegradable material, PHB has the characteristics of no toxicity, biocompatibility, biodegradability, good thermal processing performance, capability of being produced by renewable energy, high polymerization degree, high crystallinity and structural regularity of a repeating unit. Has better application potential in the aspects of preparing biodegradable plastics, biomedicine and agricultural application. P (3HB-co-4HB) is not readily hydrolyzed rapidly in the environment, but is degraded by microorganisms in the environment, can be degraded into carbon dioxide and water under aerobic conditions, and can be degraded into methane and water under anaerobic conditions.

Prothioconazole (formula 1) is a novel broad-spectrum triazolethione demethylation inhibitor bactericide, has good systemic activity, excellent treatment, protection and eradication performance, long lasting period and successful commercial development, and is in the leading position in the market of global bactericides, particularly bactericides for grains.

Prothioconazole has been registered and sold in 60 countries worldwide since its first acquisition in 2004. No registration is obtained in China before 2019, the main reason is that prothioconazole has potential harm to female reproductive systems, a metabolite desulfurization-prothioconazole generated after being sprayed to the environment has teratogenicity, the half-life period of the prothioconazole is long, and potential risks exist to the health of application personnel. The latest research finds that prothioconazole has exposure risk to aquatic organism zebra fish embryos, has certain destructive effect on gonads of male lizards, inhibits spermatogenesis in testis, and desulfurated prothioconazole has greater risk to the zebra fish. Therefore, the use of prothioconazole is limited domestically.

How to better utilize prothioconazole is a great research trend.

Disclosure of Invention

In view of the above, the present invention provides a composite antibacterial film, and a preparation method and an application thereof. The composite antibacterial film provided by the invention can prolong the lasting period of prothioconazole in soil, can effectively prevent and treat soil-borne diseases and leaf diseases in soil, can be used for plant potting and expands the application range of the prothioconazole.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a composite antibacterial film, which is cast by a mixed solution, wherein the mixed solution comprises poly (3-hydroxybutyrate-co-4-hydroxybutyrate), prothioconazole and an organic solvent;

the mass ratio of the poly (3-hydroxybutyrate-co-4-hydroxybutyrate) to the prothioconazole is (1.0-2.0): (0.01-1.0).

Preferably, the mass ratio of the poly (3-hydroxybutyrate-co-4-hydroxybutyrate) to the prothioconazole is 1.9: 0.1.

preferably, the mass content of prothioconazole in the composite antibacterial film is 1.73-37.32%.

Preferably, the mass content of prothioconazole in the composite antibacterial film is 3.96%.

Preferably, the concentration of the mixed solution is 50 mg/mL.

Preferably, the organic solvent is chloroform.

Preferably, the thickness of the composite antibacterial film is 45-55 μm.

The invention also provides a preparation method of the composite antibacterial film in the technical scheme, which comprises the following steps:

mixing poly (3-hydroxybutyrate-co-4-hydroxybutyrate), prothioconazole and an organic solvent to obtain a mixed solution;

and casting the mixed solution into a film, and obtaining the composite antibacterial film after the organic solvent is completely volatilized.

Preferably, the mixing is performed under the condition of magnetic stirring, and the time of the magnetic stirring is 1-2 hours.

The invention also provides the application of the composite antibacterial film in the technical scheme or the composite antibacterial film prepared by the preparation method in the technical scheme in the field of plant cultivation.

The invention provides a composite antibacterial film, which is cast by a mixed solution, wherein the mixed solution comprises poly (3-hydroxybutyrate-co-4-hydroxybutyrate), prothioconazole and an organic solvent; the mass ratio of the poly (3-hydroxybutyrate-co-4-hydroxybutyrate) to the prothioconazole is (1.0-2.0): (0.01-1.0). According to the invention, by reasonably controlling the mass ratio of the poly (3-hydroxybutyrate-co-4-hydroxybutyrate) and the prothioconazole in the mixed solution, the antibacterial property of the film can be ensured, the growth and development of plants are not influenced, and the application range of the prothioconazole is expanded.

Drawings

FIG. 1 is a surface morphology picture of a composite antibacterial film 1, 4-6;

FIG. 2 is a cross-sectional view of the composite antibacterial film 1, 4-6;

FIG. 3 is a histogram of the thickness of the composite antibacterial films 1-6;

FIG. 4 is a stress-strain curve of the composite antibacterial films 1-6;

FIG. 5 is a bar graph of tensile strength of the composite antibacterial films 1-6;

FIG. 6 is a bar graph of the elongation at break of the composite antibacterial films 1-6;

FIG. 7 is a histogram of Young's modulus of the composite antibacterial films 1-6;

FIG. 8 is a TG curve of the composite

antibacterial films

4 and 6 and a single PRO;

FIG. 9 is a DSC curve of the composite

antibacterial films

4 and 6 and the single PRO;

FIG. 10 is a water solubility curve of the composite antibacterial film 1-6 at different times;

FIG. 11 is an SEM electron micrograph of the composite antibacterial films 1, 4-6 after being dissolved in water for 35 days;

fig. 12 is a macroscopic photograph of the composite antibacterial film 1 before and after degradation;

FIG. 13 is an SEM picture of the composite antibacterial film after being degraded for 35 days 1, 4-5;

FIG. 14 is a graph showing the cumulative release curves of the composite antibacterial films 1 to 5 at pH 6.3;

FIG. 15 is a graph showing the cumulative release curves of the composite antibacterial films 1 to 5 at pH 7.6;

FIG. 16 is a graph showing the cumulative release curves of the composite antibacterial films 1 to 5 at pH 8.8;

FIG. 17 is an infrared spectrum of PHB, PRO and the composite antibacterial film 3-6;

FIG. 18 is a graph showing the transmittance and transparency of the composite antibacterial films 1-6, wherein A is a transmittance graph and B is a transparency graph;

FIG. 19 is a graph showing contact angles of the composite antibacterial films 1 to 6;

FIG. 20 is a graph showing the indoor bactericidal activity of the composite antibacterial films 2 to 6 and the uncovered films;

FIG. 21 is a root length chart and a fresh stem weight chart of the composite antibacterial films 1 to 4 and 6 and the uncovered films, wherein A is a root length and stem length chart; and B is fresh weight and dry weight.

Detailed Description

In the present invention, the starting materials used in the present invention are all commercially available products unless otherwise specified.

In the present invention, the mass ratio of the poly (3-hydroxybutyrate-co-4-hydroxybutyrate) (PHB) to Prothioconazole (PRO) is particularly preferably 1.95:0.05, 1.9:0.1, 1.8:0.2, 1.6:0.4 or 1.2:0.8, and more preferably 1.9: 0.1. In the present invention, the organic solvent is preferably chloroform. In the present invention, the concentration of the mixed solution is preferably 50 mg/mL.

In the present invention, the mass content of prothioconazole in the composite antibacterial film is preferably 1.73 to 37.32%, more preferably 1.73%, 3.96%, 8.75%, 18.15% or 37.32%, and still more preferably 3.96%. In the invention, the thickness of the composite antibacterial film is preferably 45-55 μm.

In the present invention, the parameters of the casting are described in detail in the preparation method section.

The invention mixes poly (3-hydroxybutyrate-co-4-hydroxybutyrate), prothioconazole and organic solvent to obtain mixed solution. In the invention, the mixing is preferably carried out under the condition of magnetic stirring, and the time of the magnetic stirring is preferably 1-2 h.

After the mixed solution is obtained, the mixed solution is cast into a film, and the composite antibacterial film is obtained after the organic solvent is completely volatilized. The casting film-forming method is not particularly limited, and a casting film-forming means known to those skilled in the art can be adopted. In the present invention, the temperature at which the organic solvent is volatilized is preferably room temperature, i.e., neither additional heating nor additional cooling is required; the time for volatilizing the organic solvent is preferably 3-5 days.

When the composite antibacterial film provided by the invention is applied to plant cultivation, the plants can not be interfered by harmful fungi and the like, and the growth and development of the plants can not be influenced.

The composite antibacterial film provided by the present invention, the preparation method and the application thereof are described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Mixing poly (3-hydroxybutyrate-co-4-hydroxybutyrate) and prothioconazole according to the mass ratio of 1.95:0.05, 1.9:0.1, 1.8:0.2, 1.6:0.4, 1.2:0.8 and 2.0:0.0, dissolving the mixture by using trichloromethane, placing the mixture on a magnetic stirrer, and stirring the mixture for 2 hours until the mixture is completely dissolved to obtain a mixed solution with the final concentration of 50 mg/mL; and respectively measuring 10mL of mixed solution by using a measuring cylinder, casting the mixed solution on the surface of a 90mm glass culture dish, placing the mixed solution in a fume hood at room temperature for 5 days until the organic solvent is completely evaporated, and finally obtaining composite antibacterial films with different prothioconazole contents, which are respectively marked as a composite antibacterial film 1, a composite antibacterial film 2, a composite antibacterial film 3, a composite antibacterial film 4, a composite antibacterial film 5 and a composite antibacterial film 6.

Performance testing

1. Determination of composite antibacterial film content by high performance liquid chromatography

Placing 0.025g (accurate to 0.0001g) of the composite antibacterial film in a 50mL volumetric flask, carrying out ultrasonic constant volume treatment with methanol to 50mL for 30min, collecting supernatant, filtering with a 0.22 μm filter membrane, and carrying out prothioconazole loading determination by using a High Performance Liquid Chromatography (HPLC). The drug loading of the composite antibacterial film is determined by adopting a formula (1):

high performance liquid chromatography conditions: the mobile phase is as follows: v (methanol): v (0.2% formic acid by volume) 80: 20, flow rate: l.0mL/min; a chromatographic column: waters sunfire-C18 chromatography column (4.6 mm. times.250 mm, 5 μm); wavelength of ultraviolet detector: 260 nm; column temperature: 30 ℃; sample introduction volume: 5 mu L of the solution; prothioconazole retention time: 7.3 min.

The drug-loading rates of prothioconazole of the obtained composite antibacterial films 1-6 are respectively 1.73%, 3.96%, 8.75%, 18.15%, 37.32% and 0%, the composite antibacterial film 6 is abbreviated as CK, and other composite antibacterial films are distinguished by the drug-loading rate of prothioconazole.

FIG. 1 is a picture of the surface morphology of the composite antibacterial film 1, 4-6, and FIG. 2 is a picture of the cross section of the composite antibacterial film 1, 4-6. As can be seen from fig. 1 and 2: in the composite antibacterial film, when the content of prothioconazole is lower than 18.15%, the blending property of PHB and PRO is better to form a uniform film, and the reason is that the PHB and PRO have better compatibility; however, when the content was 37.32%, crystals of prothioconazole precipitated.

2. Thickness measurement of composite antibacterial film

The film thickness was measured with a digital micrometer, and the results are shown in FIG. 3. As can be seen from fig. 3: the thickness of the obtained film is 45-55 mu m, and the thickness of different film contents is not obviously different.

3. Mechanical property test of composite antibacterial film

The mechanical properties of the obtained composite antibacterial film are determined according to the national standard GB/T528-2009: cutting the composite antibacterial film into a dumbbell shape, and testing the tensile strength and the elongation at break by using an electronic universal testing machine; 3 replicates were performed for each sample. Cutting the composite antibacterial film into a dumbbell shape, measuring the thickness and the width of the film by using a micrometer, recording the initial gauge length, clamping the dumbbell-shaped antibacterial film, and recording the stress-strain curve of the material at the speed of 10mm/min of stretching speed after the displacement returns to zero.

FIG. 4 is a stress-strain curve of the composite antibacterial films 1-6, FIG. 5 is a bar graph of tensile strength of the composite antibacterial films 1-6, FIG. 6 is a bar graph of elongation at break of the composite antibacterial films 1-6, and FIG. 7 is a bar graph of Young's modulus of the composite antibacterial films 1-6. From figure 4 it can be seen that: the variation trend of the composite antibacterial film in the stretching process. As can be seen from fig. 5 and 6: the tensile strength is the greatest when the PRO content is 3.96%, and then the tensile strength tends to decrease as the PRO content increases; with the increase of the PRO content, the elongation at break of the composite antibacterial film is remarkably increased by 3-8 times, and the brittleness of the PHB film is improved. The tensile strength is improved by 1.7 times when the PRO content is 3.96 percent and 8.75 percent. Fig. 7 depicts the resistance to deformation of the composite antibacterial film, and the young's modulus value of the composite antibacterial film is 356.95Pa at the maximum when the PRO content is 3.96%.

Table 1 shows the mechanical property test results of the composite antibacterial films 1-6.

TABLE 1 mechanical property test results of composite antibacterial films 1-6

Note: the different lower case letters in the same column indicate significant differences at a P <0.05 level as tested by Duncan's new double-pole-difference method.

As can be seen from table 1: the toughness of the composite antibacterial film is enhanced by adding the PRO, the tensile strength of the composite antibacterial film is increased and then reduced along with the increase of the PRO content, and the elongation at break is gradually increased. It is worth noting that: the elongation at break of the composite antibacterial film exceeds 800 percent of the pure PHB film when the proportion of PRO is 18.15 percent. Therefore, in the stretching process, the space available for chain motion in the PHB is small, and the molecular motion is very difficult; when stretched, exhibits a distinct brittle fracture characteristic; after the prothioconazole is added, the crystal regularity and continuity of PHB are destroyed, so that the crystallinity is reduced; when prothioconazole reaches a certain threshold, it will agglomerate and mix unevenly in the PHB carrier, thus resulting in a decrease in the mechanical properties of the antimicrobial film at a level of 37.32%. In conclusion, as the number of PROs continues to increase, the compatibility of the composite antibacterial film is poor, and the elastic modulus is suddenly reduced; the composite antibacterial film has enhanced flexibility and rigidity, and makes up the defects of PHB to a certain extent.

4. Analysis of thermal Properties

And performing thermodynamic performance tests on different composite antibacterial films by adopting differential scanning calorimetry and thermogravimetric analysis, and observing the change rule of corresponding thermodynamic performance indexes.

Differential Scanning Calorimetry (DSC) analysis: a differential scanning calorimeter of type Q2000 manufactured by TA of USA was used. Raising the temperature to 180 ℃ at room temperature, keeping the temperature for 10min, and eliminating the thermal history; cooling to-60 ℃, then heating to 180 ℃, and recording the second heating data; the temperature was raised in a nitrogen atmosphere at a rate of 10 ℃/min.

Thermogravimetric (TGA) analysis: a Q500 thermogravimetric analyzer produced by American TA company is adopted, the nitrogen atmosphere and the flow rate are 10mL/min, and the thermal weight loss of the composite antibacterial film in the process of increasing the temperature from room temperature to 800 ℃ by 10 ℃/min is measured.

Fig. 8 is a TG curve of the composite

antibacterial films

4 and 6 and the pure PRO, and it can be seen from fig. 8 that: the thermal decomposition stability of the composite antibacterial film 6(CK), namely a PHB film is lower than that of the prothioconazole original drug; the thermal decomposition stability of the PHB film is improved after prothioconazole is added into the PHB film. At 47.2 ℃ and 40.6 ℃, the initial weight loss of the composite antibacterial film and the PHB film is observed; whereas at 193.8 ℃, an initial weight loss of the technical prothioconazole was observed. The thermal decomposition (Td) of the composite antibacterial film exhibits a gradual weight loss process from 284.2 ℃ Td (5%) to 295.0 ℃ Td (80%); gradual weight loss process of thermal decomposition (Td) of PHB film from 284.7 ℃ to 293 ℃ Td (80%); the gradual weight loss process of PRO drug from 285.7 ℃ Td (5%) to 313 ℃ Td (80%).

Fig. 9 is a DSC curve of the composite

antibacterial films

4 and 6 and the pure PRO, and it can be seen from fig. 9 that: PHB and PRO form a stable mixture which does not separate on heating, since there is only one melting peak and one decomposition peak on the heat map. Embedding PRO in PHB lowers its melting temperature Tg by 36 deg.C, a decrease in Tg meaning an increase in melt viscosity, thereby inhibiting crystallization of poly (3-hydroxybutyrate-co-4-hydroxybutyrate); therefore, small crystals are formed in the composite antibacterial film, and the crystals begin to melt at a lower temperature than the initial polymer, so that the melting temperature of the mixture is reduced, and the structure of the poly (3-hydroxybutyrate-co-4-hydroxybutyrate) is more amorphous.

5. Water solubility test and observation of degradation form of composite antibacterial film in soil

Drying 0.3g of composite antibacterial film with different drug loading rates at 100 ℃ for 2h, and recording the initial mass M₀. And respectively placing the composite antibacterial films in 50mL of distilled water at room temperature. After different time intervals, the solution is removed, and the composite antibacterial film is taken out and dried in a vacuum drying oven at 100 ℃ for 2 hours. The final masses Mt are recorded separately. The water solubility is calculated according to the following formula:

and (3) dissolving and degrading the composite antibacterial film in water for 35 days, taking out and drying, and observing the film shape by using an SEM (scanning electron microscope).

Cutting the composite antibacterial film into a square with the size of 3cm multiplied by 3cm, and burying the square in soil (200 g); after 1 month, the sample was removed, washed with distilled water, dried in an oven at 100 ℃ for 2 hours, and removed under SEM electron microscope to observe the change in morphology.

FIG. 10 is a water solubility curve of the composite antibacterial films 1-6 at different times, and FIG. 11 is an SEM electron micrograph of the composite antibacterial films 1, 4-6 after being dissolved in water for 35 days. As can be seen from fig. 10 and 11: the CK, namely PHB film cannot be dissolved in water, and the water solubility of the composite antibacterial film is gradually increased along with the increase of the PRO drug loading and the prolonging of time. The drug loading rate after 35 days is 1.73%, 3.96%, 8.75%, 18.15% and 37.32%; the mass loss rate of the composite antibacterial film in sterile water is 0.82%, 2.38%, 4.53%, 9.61% and 6.74%, respectively. With the increase of the drug loading of the composite antibacterial film, the mass loss rate is gradually increased, but when the drug loading rate is 37.32%, the mass loss rate is lower than that of the film with the drug loading rate of 18.15%. For the reason, when the content of prothioconazole in the composite antibacterial film is high, prothioconazole is separated out in water in a crystallization mode; the prothioconazole is not separated out when the prothioconazole is dissolved in water to saturation. SEM micrograph shows that: when the PRO content is low, fine cracks appear on the surface of the composite antibacterial film and become uneven, and when the PRO content is high, the surface of the composite antibacterial film becomes less uneven than the composite antibacterial film having a low PRO content, and thus, it was also confirmed that the mass loss rate of the antibacterial film having the PRO content of 37.32% in water is slightly lower than that of the composite antibacterial film having the PRO content of 18.15%.

Fig. 12 is a macro photograph of the composite antibacterial film 1 before and after degradation, and fig. 13 is an SEM picture of the composite antibacterial film 1 after degradation for 35 days from 4 to 5 days. As can be seen from fig. 12 and 13: the composite antibacterial film with different drug-loading rates is degraded after one month in soil, and the surface of the composite antibacterial film becomes rough, has holes and cracks along with the extension of soil burying time. The composite antibacterial film is a biodegradable material, and the degradation of the composite antibacterial film in soil is greatly influenced by the number of environmental microorganisms, the environmental temperature, the humidity and the pH value.

6. Slow release performance of composite antibacterial film in release media with different pH values

In order to research the slow release performance of pesticide molecules PRO in the composite antibacterial films with different drug-loading rates, three release media with different pH values are prepared to measure the release performance, and the release media are prepared from three components, namely Phosphate Buffer Solution (PBS), absolute ethyl alcohol and tween-80 according to the volume ratio of 70: 29.5: 0.5, and mixing. The 20mg composite antibacterial film is placed in a plastic bottle with a cover and containing 100mL release medium, the plastic bottle is fixed on a shaker, the plastic bottle is shaken at 150rpm under the condition of 25 ℃, and 0.6mL of release medium is taken out at certain time intervals for HPLC analysis. Three replicates of each experiment were performed. The release rate of prothioconazole in the composite antibacterial film is calculated according to the following formula:

er: cumulative released amount (%); ve: volume sampled per time (0.6 mL); c_i: concentration of released solution (mg/mL) at the time of ith sampling; v₀: total release medium volume (100 mL); cn: the concentration (mg/mL) of the released solution in the nth sampling; n: sampling times; m is_pesticide: total mass (mg) of active ingredients of PRO/PHB film.

Fig. 14 is a cumulative release curve of the composite antibacterial films 1 to 5 at pH 6.3, fig. 15 is a cumulative release curve of the composite antibacterial films 1 to 5 at pH 7.6, and fig. 16 is a cumulative release curve of the composite antibacterial films 1 to 5 at pH 8.8; Ritger-Peppas fitting was performed on the release curve, and the results are shown in Table 2.

TABLE 2 Release Curve Ritger-Peppas fitting results

The logarithmic form of the Ritger-Peppas equation is: log (M)_t/M_∞) ═ logk + nlogt, where M_t/M_∞For the cumulative percent drug release, k is the drug release rate constant and n is the diffusion index. The type of release mechanism can be inferred from the value of n: when n is less than or equal to 0.45, the drug release is mainly Fick diffusion; when 0.45<n<At 0.89, the release is mainly non-Fick diffusion, and the drug release is in coexistence of diffusion and erosion; when n is>At 0.89, the drug is released mainly by erosion of the skeleton of the drug-carrying system. The composite antibacterial film is dissolved in a buffer medium, the release rule is mainly corrosion, the prothioconazole is released in two processes of firstly 'burst release' and then 'slow release', the 'burst release' rate is higher than the 'slow release' rate, and the release of the composite antibacterial film is mainly Fick diffusion. When the content of the composite antibacterial film PRO is 1.73% and the pH value is 7.6, the release process is mainly non-Fick diffusion, and the drug release is diffused in erosion and exists. The release data of the experiment are simulated to show that the release responsiveness of the composite antibacterial film in solutions with different pH values is not large.

7. Attenuated total reflectance Fourier transform Infrared Spectroscopy (ATR-FTIR) testing

And testing the surface characteristics of the composite antibacterial film with different drug-loading rates by adopting ATR-FTIR (attenuated reflectance-infrared spectroscopy) to evaluate the crystallization performance of the composite antibacterial film. Setting resolution to 4cm^-1Wavelength of 400-4000 cm^-1The number of scans was 64.

FIG. 17 is an infrared spectrum of PHB, PRO and the composite antibacterial film 3-6. As can be seen from fig. 17: 1720cm^-1The position of the C-O stretching vibration corresponds to the carbonyl group in the molecular structure of PHB, and the C-H stretching vibration exists in the PHB, so that the length of 2978cm is displayed^-1Left and right main peaks; the prothioconazole is in the concentration of 3000cm^-1The position of the absorption peak appears as an asymmetric telescopic vibration absorption peak of a C-H bond of a benzene ring, 1552cm^-1750cm for the stretching and the vibration of the benzene ring framework^-1Stretching vibration of C-Cl bond; with the increase of the content of prothioconazole, the strength of the chemical bond is gradually increased. In addition, no new characteristic peak appears when prothioconazole is added into PHB, which indicates that the two are simply and physically mixed and are not combined by chemical bonds.

8. Measurement of light transmittance

Cutting the composite antibacterial film 1-6 into long-strip-shaped samples with the length of 3cm and the width of 1cm, measuring the light transmittance of the composite antibacterial film by adopting an ultraviolet-spectrophotometer, selecting the wavelength range from 200 nm to 1000nm, measuring each sample for 3 times, and calculating the average value.

Finally, the transparency value at 600nm is calculated according to the following formula:

in the formula, T₆₀₀The transmittance at 600nm is, and x is the film thickness (mm). A higher T value (Transparency value (%)) indicates that the film is less transparent.

FIG. 18 is a graph showing the transmittance and transparency of the composite antibacterial films 1-6, wherein A is a transmittance graph and B is a transparency graph. There are various environmental factors affecting the growth and development of crops, and the temperature has the greatest influence on the growth and development of crops. Soil temperature has a significant impact on the propagation and yield of crops. Mulching film is widely used in agriculture because it can affect soil heat and soil temperature; the transparent mulching film has a better soil heat preservation effect than other mulching films due to the excellent light transmittance. Therefore, the research on the transparency of the composite antibacterial film is of great significance. As shown in A in FIG. 18, the light transmittance can be 60% or more in the range of 400 to 1000 nm. The composite antibacterial film with prothioconazole as a light yellow substance and PRO addition amount of 37.32% has small change of light transmittance, thereby showing that: although the color of prothioconazole itself has less effect on the light transmittance of the film, because PRO is at higher concentration, PHB crystallization rate is increased. The composite antibacterial film SEM shows that: when the content of PRO is high, the crystallization performance of the composite antibacterial film is improved; the transmittance thereof is reduced. Panel B in fig. 18 shows: the lower the PRO content of the drug-loaded thin film with different prothioconazole contents is, the higher the light transmittance is; conversely, the higher the PRO content, the lower the light transmittance. When the content of the prothioconazole is less than 20 percent, the influence of the content on the transmittance of the composite antibacterial film is not obvious.

9. Measurement of contact angle of composite antibacterial film

The contact angle measuring instrument can clearly display the image of a water drop on a computer screen, the data of the contact angle of the water drop is read for 1-10 min through system software, fig. 19 is a contact angle graph of the composite antibacterial film 1-6, and as can be seen from fig. 19: the composite antibacterial films are all hydrophobic films, and no significant difference exists between contact angles.

10. Antimicrobial Activity detection

In vitro antibacterial activity: the southern blight of the peanut is selected as a test strain, the composite antibacterial film is cut into squares of 5 multiplied by 5mm, and the bactericidal activity of the composite antibacterial film on the southern blight pathogenic bacteria is measured by adopting a hypha growth rate method. Inoculating 4mm of the cultured peanut southern blight pathogenic bacteria cake to the center of a PDA (personal digital assistant) plate, placing a 5 multiplied by 5mm square composite antibacterial film around the peanut southern blight pathogenic bacteria cake, and observing the antibacterial activity of the peanut southern blight pathogenic bacteria cake on bacterial colonies.

FIG. 20 shows the indoor bactericidal activity of the composite antibacterial films 2 to 6 and the uncoated film, wherein CK-represents the uncoated film. As can be seen from fig. 20: the blank PHB film, namely the composite antibacterial film 6 has no antibacterial activity on pathogenic bacteria of the southern blight and has no difference with the CK film. But the antibacterial performance of the composite antibacterial film is gradually enhanced along with the increase of the content of the prothioconazole. It proves that: the drug-containing composite antibacterial film has certain inhibitory activity on southern blight.

11. Pot experiment

The method comprises selecting full, undamaged and uniform peanut seeds for pot experiment, sterilizing with 0.1% (v/v) sodium hypochlorite water solution for 3min before sowing, and washing with sterile water for 3 times. The peanut seeds are disinfected and germinated by a culture dish filter paper method, and seedlings with consistent growth vigor are selected and transplanted into a seedling raising pot after the germs break through the seed coats by 1 cm. The seedling raising soil is from a land block where perennial peanuts are continuously planted and soil-borne diseases are serious. Taking the soil with the soil layer depth of 5-20 cm back to a laboratory, removing plant residues, stones and the like in the soil, and then fully and uniformly mixing the soil by using a sieve with the aperture of 2 mm. They were cultivated in a greenhouse and equal amounts of water were poured into each pot daily to maintain suitable growth conditions. Two weeks after sowing, the root length, stem length, fresh weight and dry weight of the peanut seedlings were measured.

Collecting soil in a land where peanuts are continuously planted in a field all year round and soil-borne diseases are serious, planting the peanuts in a greenhouse flowerpot, covering soil with a composite antibacterial film, observing the flower growth vigor 17 days after the emergence of the peanuts, and measuring the root length, stem length, fresh weight and dry weight of the peanuts.

FIG. 21 is a root length chart and a fresh stem weight chart of the composite antibacterial films 1 to 4 and 6 and the uncovered films, wherein A is a root length and stem length chart; b is fresh weight and dry weight; as can be seen from the pot experiment: the growth vigor of the control of the uncovered film is generally weaker, and the growth vigor of the peanut seedlings covered with the composite antibacterial film 6 is better than that of the peanut seedlings not covered with the film. With the rising of the PRO content of the composite antibacterial film, the root length, fresh weight and dry weight of the peanut seedlings show the trend of increasing first and then decreasing; stem length data show: the composite antibacterial film 6, namely the PHB blank film, has the longest stem length, and the lowest stem length when the PRO content is 18.15 percent. The pot experiment data show that the growth of pathogenic bacteria is inhibited under the low concentration of PRO in the composite antibacterial film, so that the growth of peanuts is promoted, and the optimal concentration of the composite antibacterial film is 3.96 percent; the peanut with high PRO content in the composite antibacterial film has weaker growth potential, and the analysis reason is that: the high-content PRO antibacterial film may cause phytotoxicity to peanut seedlings, so that the peanut seedlings are weak in growth vigor.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. The composite antibacterial film is formed by casting a mixed solution, and is characterized in that the mixed solution comprises poly (3-hydroxybutyrate-co-4-hydroxybutyrate), prothioconazole and an organic solvent;

the mass ratio of the poly (3-hydroxybutyrate-co-4-hydroxybutyrate) to the prothioconazole is (1.0-2.0): (0.01 to 1.0);

the mass content of prothioconazole in the composite antibacterial film is 1.73-8.75%.

2. The composite antibacterial film according to claim 1, wherein the mass ratio of the poly (3-hydroxybutyrate-co-4-hydroxybutyrate) to prothioconazole is 1.9: 0.1.

3. the composite antibacterial film according to claim 1, wherein the mass content of prothioconazole in the composite antibacterial film is 3.96%.

4. The composite antibacterial film according to claim 1, wherein the concentration of the mixed solution is 50 mg/mL.

5. The composite antibacterial film according to claim 1, wherein the organic solvent is chloroform.

6. The composite antibacterial film according to claim 1, wherein the composite antibacterial film has a thickness of 45 to 55 μm.

7. A method for preparing a composite antibacterial film according to any one of claims 1 to 6, characterized by comprising the steps of:

8. The preparation method of claim 7, wherein the mixing is performed under the condition of magnetic stirring, and the time of the magnetic stirring is 1-2 h.

9. Use of the composite antibacterial film according to any one of claims 1 to 6 or the composite antibacterial film prepared by the preparation method according to any one of claims 7 to 8 in the field of plant cultivation.