CN114766477A

CN114766477A - Application of polyethyleneimine-cellulose composite gel

Info

Publication number: CN114766477A
Application number: CN202210507722.0A
Authority: CN
Inventors: 孟琴; 沈冲; 吴升冬; 卢丹; 肖海峰
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-05-10
Filing date: 2022-05-10
Publication date: 2022-07-22
Anticipated expiration: 2042-05-10
Also published as: CN114766477B

Abstract

The invention discloses an application of polyethyleneimine-cellulose composite gel, which mainly comprises the steps of killing rice blast fungus (Magnaporthe grisea), melon vine fungi (Mycosphaerella melonis), Penicillium fungi (Penicillium), Alternaria alternata (Alternaria), Pythium sp, Saccharomyces cerevisiae (Saccharomyces cerevisiae) and the like. The composite gel can prevent and treat rice blast and gummy stem blight of melons, and can also be used for preventing fruit and vegetable rot caused by penicillium, alternaria, pythium and saccharomyces cerevisiae. The composition of said polyethylene imine-cellulose composite gel includes 1-10 wt% of cellulose derivative (i.e. oxidized cellulose and carboxymethyl cellulose) and 1-10 wt% of polyethylene imine. Both are prepared by one of amidation reaction and Schiff base reaction. Because the plant bactericide has almost no toxicity to animal cells, the plant bactericide has better human safety when being applied to plant antiseptics and fruit and vegetable preservatives.

Description

Application of polyethyleneimine-cellulose composite gel

Technical Field

The invention relates to an application of polyethyleneimine-cellulose composite gel, belonging to the application field of antibacterial agents.

Background

Cellulose is a widely used natural biomass, and is considered as an inexhaustible natural chemical raw material due to its relative environmental friendliness and excellent biocompatibility. However, due to the property of being insoluble in water, the use scene is limited. Cellulose-based hydrogels have been favored by many researchers because they are made into hydrogels to impart water dispersibility, surface activity, etc.

Polyethyleneimine (PEI) is a polycationic antibacterial agent, which is polymerized from cyclic ethylamine and is an organic macromolecule with high positive charge content, and the molecular structure of the polyethyleneimine can be divided into linear polyethyleneimine (L-PEI) and branched polyethyleneimine (B-PEI). PEI has a protonated amino nitrogen atom for every two carbon atoms apart (Thomas M et al, Proc Natl Acad Sci US A.2005,102: 5679-: it carries a large amount of positive charges on its surface, targets negatively charged proteins on bacterial cell membranes, and when adsorbed to the cell surface by electrostatic attraction, branches insert into the bacterial cell membrane, rupturing the membrane and releasing membrane contents leading to cell death (Ghamrawi S et al, Mater Sci Eng C Mater Biol Appl,2017,5: 69-979). In order to improve the biocompatibility of PEI, PEI is modified or chemically bonded with other substances with good biocompatibility to prepare an antibacterial compound with low cytotoxicity and good antibacterial activity (Shevchenko V D et al, Mater Sci Eng C Mater Biol Appl,2016,69: 60-67).

The PEI and the cellulose are compounded to prepare the hydrogel, and the hydrogel is expected to have potential application in the antibacterial aspect of foods and crops.

Disclosure of Invention

The invention aims to combine polyethyleneimine and cellulose and provides application of polyethyleneimine and cellulose composite gel in the fields of fruit and vegetable corrosion prevention, plant disease control and the like.

The technical scheme adopted by the invention is as follows:

use of a polyethyleneimine-cellulose composite gel, comprising:

a. killing one or more of Magnaporthe grisea, Rhizoctonia solani (Mycosphaerella melonis), Penicillium (Penicillium), Alternaria (Alternaria), Pythium sp, and Saccharomyces cerevisiae (Saccharomyces cerevisiae);

b. preventing and treating rice blast and/or melon gummy stem blight;

c. for preventing or slowing the decay of fruits and/or vegetables caused by penicillium, alternaria, pythium and/or saccharomyces cerevisiae.

2. The use according to claim 1, wherein the composition of the polyethyleneimine-cellulose composite gel comprises 1 to 10 wt% of cellulose derivatives (oxidized cellulose, carboxymethyl cellulose and the like), 1 to 10 wt% of polyethyleneimine, and the balance of water;

further, the cellulose derivative is one or two of oxidized cellulose and carboxymethyl cellulose;

further, the molecular weight of the polyethyleneimine is 5000-70000 Da.

Further, the polyethyleneimine-cellulose composite gel can be prepared by one of amidation reaction and schiff base reaction according to different cellulose derivatives. The method specifically comprises the following steps: aldehyde group in the oxidized cellulose and amino group in the polyethyleneimine are subjected to Schiff base reaction to generate hydrogel; or the carboxyl in the carboxymethyl cellulose and the amino in the polyethyleneimine have amidation reaction to generate hydrogel; the combination with cellulose can improve the biocompatibility of polyethyleneimine.

Further, the method for preparing the polyethyleneimine-cellulose composite gel by Schiff base reaction comprises the following steps:

injecting the polyethyleneimine solution and the oxidized cellulose solution into a cylindrical container to obtain the hydrogel. The concentration of the two solutions is 1-10 wt%, the mass ratio of the polyethyleneimine to the oxidized cellulose is 0.5:1-5:1, the pH value is 6-10, and the molecular weight of the polyethyleneimine is 5000-; preferably, the concentration of the two solutions is 3-5 wt%, the mass ratio of the polyethyleneimine to the oxidized cellulose is 1:1-2:1, the pH is 7-9, and the molecular weight of the polyethyleneimine is 10000-70000 Da.

Further, the method for preparing the polyethyleneimine-cellulose composite gel through amidation reaction is as follows:

the polyethyleneimine solution and the carboxymethylcellulose solution were injected into a cylindrical container in equal volumes, 0.1 wt% of N-hydroxysuccinimide (NHS) and 0.1 wt% of carbodiimide (EDC) were added, mixed well and incubated at 37 ℃ for 2 hours to obtain a hydrogel. The concentration of the two solutions is 1-10 wt%, the mass ratio of the polyethyleneimine to the carboxymethyl cellulose is 0.5:1-5:1, the pH value is 4-8, and the molecular weight of the polyethyleneimine is 5000-; preferably, the concentration of the two solutions is 3-5 wt%, the mass ratio of the polyethyleneimine to the carboxymethyl cellulose is 1:1-2:1, the pH is 6-7, and the molecular weight of the polyethyleneimine is 10000-70000 Da.

Further, before use, the hydrogel is placed in water and is broken up by a high-speed homogenizer to prepare a gel colloidal solution. Hydrogel: the mass ratio of the water is 1:1-1:10, preferably 1:2-1: 5.

The gel obtained by the preparation method has the beneficial effect of efficiently killing yeast and mould, and has almost no toxicity to mammalian cells and human skin. Due to the special beneficial effects, the polyethyleneimine-cellulose composite gel prepared by the invention can be used as a fruit/vegetable preservative to inhibit the rot action of mould and yeast on fruits and vegetables, so that the fruits and vegetables can be preserved for a long time. In addition, the composite gel can efficiently kill rice blast (Magnaporthe grisea) and melon vine blight (Mycosphaerella melonis), and shows good effects of preventing and treating rice blast and melon vine blight on plant experiments.

Drawings

FIG. 1 is a graph of preparation conditions (a. charge ratio, b. oxidation reaction time, c.pH) of oxidized cellulose versus infrared characterization (d);

FIG. 2 is a diagram of a polyethyleneimine-cellulose composite gel bacteriostatic circle (a. Escherichia coli, b. Staphylococcus aureus);

FIG. 3 is a graph showing the results of the evaluation of the polyethyleneimine-cellulose complex gel for skin safety;

FIG. 4 is a graph showing the effect of a polyethyleneimine-cellulose complex gel on inhibiting Pyricularia oryzae (a) and Rhizoctonia solani (b)

Detailed Description

Example 1: preparation of oxidized cellulose

100ml of a 70 wt% sulfuric acid aqueous solution was prepared, 10g of corncob cellulose was put into the sulfuric acid aqueous solution, stirred until the solution became transparent (about 30 minutes), diluted with 10-fold water and centrifuged, and then the supernatant was removed, and the hydrolyzed cellulose was collected and washed with water. Sampling and drying, and determining the yield of the hydrolyzed cellulose to be 55 percent according to a gravimetric method. Taking cellulose hydrolysate, diluting the cellulose hydrolysate to 5% (w/w), adding sodium periodate, reacting for 1-7h, with the pH value of 2-8, and the feeding ratio: 0.5:1-5:1, adding ethylene glycol to stop the reaction, taking out, dialyzing, freeze-drying and storing to obtain the oxidized cellulose.

The relationship between the three reaction conditions of the charge ratio (reaction time 3h, pH 6), the oxidation reaction time (pH 4, charge ratio 1.5: 1) and the pH (reaction time 3h, charge ratio 1.5: 1) and the aldehyde content and yield is shown in FIG. 1, and the optimal reaction conditions are shown by measuring the aldehyde content and the reaction yield: the reaction time was 3h, the pH was 4.6, the charge ratio was 1.5: under the conditions, oxidized cellulose with a yield of 82.3% and an aldehyde group content of 4.23mmol/g was obtained. The obtained oxidized cellulose solution is characterized by an infrared spectrometer, a particle size analyzer, a scanning electron microscope and the like. From its IR spectrum (lower right of FIG. 1), it was found that the oxidized cellulose was at 1733cm^-1A distinct absorption peak appears at the position generated by C ═ O stretching vibration, indicating that the cellulose has been successfully oxidized.

Example 2: preparation of polyethyleneimine-cellulose composite gel

A5 wt% polyethyleneimine (molecular weight 70000Da, pH 7) solution was mixed with 5 wt% oxidized cellulose (pH 7) in volume ratios of 0.5:1, 1:1, 1.5:1, and 2:1, and injected into a PDMS mold hole having a hole diameter of 9mm and a height of 6mm to form a hydrogel. All groups are gelatinized within 1 minute, the gel is placed for 12 hours until the gel is fully gelatinized, the hydrogel is taken out and is soaked in sterile water for balancing.

Example 3: preparation of polyethyleneimine-cellulose composite gel

A10 wt% polyethyleneimine (molecular weight: 10000Da, pH 7) solution was mixed with 3 wt% carboxymethylcellulose (pH 7) in volume ratios of 0.5:1, 1:1, 1.5:1, and 2:1, 0.1 wt% N-hydroxysuccinimide (NHS) and 0.1 wt% carbodiimide (EDC) were added, and injected into a PDMS mold hole having a hole diameter of 9mm and a height of 6mm to form a hydrogel. All groups had gelled after 12h at room temperature, after which the hydrogels were removed and equilibrated by immersion in sterile water.

Example 4: antibacterial property of polyethyleneimine-cellulose composite hydrogel

Escherichia coli (E.coli) and Staphylococcus aureus (S.aureus) cultured in LB liquid medium were each diluted to OD with physiological saline ₆₀₀1, the bacterial concentration at this time is 10⁹CFU/ml. Diluting it with physiological saline to 10⁴After CFU/ml, 1ml of the hydrogel was applied to LB solid medium, the hydrogel prepared in example 2 was placed on the solid medium coated with bacteria, and the growth of the bacteria was observed after culturing at 37 ℃ for 24 hours. As can be seen from fig. 2, when the ratio of polyethyleneimine: the mass ratio of the oxidized cellulose is from 0.5:1 to 1.5:1, the radius of the zone of inhibition becomes large, and the ratio of polyethyleneimine: the mass ratio of the oxidized cellulose is 2: the radius of the inhibition zone at 1 hour is 1.5:1 is almost the same. Therefore, the composite hydrogel has good antibacterial activity.

The minimum inhibitory concentration of the polyethyleneimine-cellulose composite hydrogel on 4 types of eukaryotic cells (Saccharomyces cerevisiae, Penicillium, Alternaria alternata Alternaria and Pythium sp, all purchased from ATCC) which commonly cause fruit rot is determined by a double dilution method. The results are shown in the following table. Therefore, the composite hydrogel has obvious inhibition effect on both saccharomycetes and mould.

TABLE 1 Minimum Inhibitory Concentration (MIC) of polyethyleneimine-cellulose composite hydrogel for 4 kinds of eukaryotic cells

Example 5: preparation of polyethyleneimine-cellulose composite gel solution

The polyethyleneimine-cellulose composite hydrogel with the solid content of 6 wt% is put into a certain amount of distilled water, and is homogenized for five minutes at 35000rpm/min by a handheld homogenizer (8mm cutter head), so as to obtain a polyethyleneimine-cellulose composite gel solution. A sample was taken, dried and weighed, and distilled water was added to give a final gel concentration of 0.4 wt%.

The gel solution is slightly turbid milky white, and the average diameter of the gel solution is 2.3 mu m through the test of a nanometer particle sizer. After standing at room temperature for 30 days, it was found that no delamination or precipitation occurred. The preparation belongs to colloidal solution and has better stability.

Example 6: cytotoxicity test of polyethyleneimine-cellulose composite gel

Culturing human fibroblast and human umbilical vein endothelial cell (all purchased from ATCC) on 24-well cell culture plate, respectively, injecting 1ml DMEM medium, placing the culture plate at 37 deg.C, and containing 5% CO₂The incubator of (1) was incubated for 24 h. Subsequently, polyethyleneimine-cellulose complex gel at a final concentration of 0.4 wt%, or 0.4 wt% polyethyleneimine (molecular weight 70000Da) was added to the medium, and incubation was continued for 24 hours, and the cell viability was measured by the thiazole blue (MTT) method. The cell viability of the gel-added group and the polyethyleneimine-added group was calculated by taking the viability of the control group (without adding complex gel or polyethyleneimine) as 100%. The results are shown in Table 2. It can be seen that 0.4% polyethyleneimine-cellulose complex gel has little cytotoxicity, while polyethyleneimine causes severe cytotoxicity. Therefore, after the polyethyleneimine and the cellulose are compounded, the cytotoxicity can be greatly reduced.

TABLE 2 cytotoxicity testing of polyethyleneimine-cellulose composite gels

Example 7: skin toxicity testing of polyethyleneimine-cellulose composite gels

Fresh pig skin was taken from the back area of female pigs supplied from a slaughterhouse, washed and trimmed of hair follicles and subcutaneous fat, and cut into uniform small pieces (+ -10X 15 mm). The epidermis was covered with a non-woven fabric impregnated with distilled water, a non-woven fabric impregnated with 0.4% (w/w) and 6% (w/w) of gel, respectively, and the effect of the gel on the growth of pig skin was observed. The pigskin was cultured in a sterile Transwell (. PHI.12 mm), and air-cultured using a medium obtained by mixing DMEM and KSFM medium in equal amounts. The culture plate was placed at 37 ℃ and 5% CO₂The culture medium is replaced every 24 hours.

After 48h of incubation, the pigskin was removed and fixed with paraformaldehyde overnight, embedded, snap frozen with liquid nitrogen and sectioned in a microtome with a thickness of 15 μm. The sliced pigskin was fixed with tissue adhesive slides and stained with H & E. As a result, as shown in FIG. 3, the fresh pig skin was intact and continuous in the horny layer, thick in thickness, and distinct in the layers between the horny layer, epidermis and dermis, and exhibited a normal healthy appearance. After two days of incubation, the control group without added gel was almost identical to the 0.4% gel treated pigskin sample. While the 6% high concentration gel resulted in a slight contraction of the cristae grooves at the true epidermal interface, no significant toxicity was seen.

Example 8: inhibitory Effect of polyethyleneimine-cellulose composite gel on Pyricularia oryzae and Rhizoctonia solani

Preparing PDA culture medium, and high-temperature sterilizing. After the sterilized PDA medium was cooled to 50-60 deg.C, the complex gel solution prepared in example 5 was added to adjust the gel concentrations to 0.1, 0.05 and 0.025 g/L. The medium containing mycelial bacteria (Mycosphaerella mellonis, purchased from ATCC) and Magnaporthegrisea (purchased from ATCC) was cut with a sterilized blade, and the medium containing mycelial bacteria was placed in a small square on the medium mixed with the complex gel and cultured in a constant temperature incubator at 28 ℃ for 7 to 14 days.

As a result, as shown in FIG. 4, the composite gel of three concentrations was effective in inhibiting the growth of Rhizoctonia solani, while the gels of two concentrations, 0.1 and 0.05g/L, were effective in inhibiting the growth of Pyricularia oryzae.

Example 9: prevention and treatment effect of polyethyleneimine-cellulose composite gel on sweet melon seedlings infected by rhizoctonia solani

And (4) inoculating the goat horn honey seedlings with consistent growth vigor. Two inoculation methods are adopted: one is to scratch the root in the substrate with a knife before inoculation, and then use a suspension of spores of Ralstonia sorghii (10% concentration)⁶seed/mL) cotton is applied to the root injury; secondly, the concentration is directly 10⁶Each/mL of the suspension of the spores of the Ralstonia solanacearum is sprayed on the leaf surfaces of seedlings and in soil, and the spraying amount of each plant is 20 mL. Plaque appeared on the plants 27 days after inoculation.

24 infected plants with plaque were selected and divided into two groups of 12 plants each, one group was dosed without any treatment and 12 normal plants were used as control groups. After the concentration of the composite gel was adjusted to 0.05g/L using the composite gel solution prepared in example 5, the diluted hydrogel was uniformly sprayed on the infected leaf surfaces of the administration group once a day. The plants were continuously recorded and observed for growth. After one month of plant cultivation, the results are reported in the following table (wherein the withered and yellow leaf rate and the number of results are only calculated for the surviving plants; the withered and yellow leaf rate is the total number of leaves with withered and yellow area > 1/3/total number of leaves x 100%; the number of results is the number of fruits with diameter larger than 5 cm). Therefore, after the composite gel is sprayed, the infection of the rhizoctonia on the sheep horn honey plants can be effectively controlled, and the survival rate and the fruiting rate of the plants are improved.

TABLE 4 prevention and treatment effects of polyethyleneimine-cellulose composite gel on cranberry-infected sweet melon seedlings

Example 10: prevention and treatment effect of polyethyleneimine-cellulose composite gel on rice blast pathogen infected rice plants

Selecting 100 pre-heading rice plants with consistent growth potential, and using the rice plants with the concentration of 10⁶The rice blast germ spore suspension is sprayed on the leaf surface and the soilThe spraying amount of each plant in the soil is 20 ml. Plaque appeared on the plants 14 days after inoculation.

50 infected plants with plaque were selected, divided into 25 plants each, one group was administered without any treatment, and 25 normal plants were used as a control group. After adjusting the gel concentration to 0.1g/L, the diluted hydrogel was uniformly sprayed on the infected leaf surfaces of the dosing groups once a day. Continuously recording and observing the growth condition of the plants. After 45 days of plant culture, the results are reported in the following table (wherein the leaf withering and yellowing rate and the rice quality are calculated only for the surviving plants, and the leaf withering and yellowing rate is calculated as in example 9). Therefore, after the gel is sprayed, the infection of rice blast germs can be effectively controlled, and the survival rate and the heading rate of plants are improved.

TABLE 5 prevention and treatment effects of polyethyleneimine-cellulose composite gel on rice blast infection of rice plants

Example 11: testing performance of polyethyleneimine-cellulose composite gel for fruit and vegetable fresh-keeping and corrosion-prevention spraying

Selecting 60 tomatoes with similar sizes and no obvious trauma (purchased from local farmer markets), spraying 75% ethanol on the surface, naturally air drying, and dividing into 3 groups (20 in each group): the experimental groups were sprayed with 10⁷CFU/ml Alternaria (Alternaria, available from ATCC) spore solution 3ml, air-dried, and sprayed with 3ml gel solution 0.5 mg/ml; positive control group was sprayed with 10 alone⁷CFU/ml Alternaria alternata spore solution 3ml, spraying sterile water 3ml to the negative control group, independently placing each small tomato in a preservation box and isolating each other, placing in an incubator at 37 ℃, and recording the number of rotten tomatoes every day. The same procedure was used for fresh-keeping experiments on strawberries (purchased from local strawberry farms) using Pythium sp (from ATCC). The results are shown in tables 6 and 7.

TABLE 6 number of rotten tomatoes

TABLE 7 number of rotten strawberries

The results show that the positive control group inoculated with alternaria alternate decays at the first day, all decays at the fourth day, and only two of the small tomatoes in the experimental group do not decay in the first four days and decay in the fifth day, which indicates that the gel solution has a remarkable preservative effect on the small tomatoes. Similarly, the results of the strawberry group also demonstrated that the spray gel delayed decay, which served the purpose of keeping the fruit fresh.

Example 12: testing performance of polyethyleneimine-cellulose composite gel as fruit fresh-keeping and corrosion-preventing spray

60 Kyoho grapes with similar sizes and without obvious trauma (purchased from local farmer markets) are selected, sprayed on the surface by using 75% ethanol, and after being naturally dried, are divided into 3 groups (20 in each group): the experimental group was sprayed with 10⁷3ml of a CFU/ml solution of Saccharomyces cerevisiae (available from ATCC) was sprayed with 3ml of a 0.5mg/ml gel solution after air-drying; positive control group was sprayed with 10 spray alone⁷3ml of the CFU/ml saccharomyces cerevisiae solution, 3ml of sterile water for the negative control group, and placing each grape in a preservation box separately and mutually isolated, placing in an incubator at 37 ℃, and recording the number of rottenness every day.

The same procedure was used to perform a fresh-keeping experiment on citrus (purchased from local farmer markets, Taizhou, Zhejiang) using Penicillium (purchased from ATCC). The results are shown in tables 8 and 9.

TABLE 8 number of rotten grapes

TABLE 9 number of rotten oranges

According to the results, the positive control group inoculated with the saccharomyces cerevisiae starts to rot in the first day, all rot occurs in the fourth day, no rot occurs in the first 2 days in the experimental group, and only 5 rot occur in the fifth day, which indicates that the gel solution has a remarkable preservative effect on grape preservation. Similarly, the results of the citrus group also demonstrated that spraying the gel delayed the decay and achieved the freshness of the fruit.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This need not be, nor should all embodiments be exhaustive. And obvious variations or modifications of the invention may be made without departing from the scope of the invention.

Claims

1. The use of a polyethyleneimine-cellulose composite gel, which comprises:

a. killing one or more of Magnaporthe grisea, Rhizopus niveus (Mycosphaerella melonis), Penicillium (Penicillium), Alternaria (Alternaria), Pythium (Pythium sp), Saccharomyces cerevisiae (Saccharomyces cerevisiae);

b. preventing and treating rice blast and/or gummy stem blight of melons;

2. The use according to claim 1, wherein the composition of the polyethyleneimine-cellulose composite gel comprises 1 to 10 wt% of a cellulose derivative, 1 to 10 wt% of polyethyleneimine, and the balance of water.

3. Use according to claim 2, characterized in that the cellulose derivative is one or both of oxidized cellulose and carboxymethyl cellulose.

4. Use according to claim 2, characterized in that the polyethyleneimine has a molecular weight of 5000-70000 Da.

5. The use according to claim 1, wherein the polyethyleneimine-cellulose composite gel is prepared by one of an amidation reaction and a schiff base reaction.

6. The use as claimed in claim 5, wherein the polyethyleneimine-cellulose composite gel is prepared by amidation reaction, and the preparation process is as follows:

mixing a polyethyleneimine solution and a carboxymethyl cellulose solution, adding 0.1 wt% of N-hydroxysuccinimide (NHS) and 0.1 wt% of carbodiimide (EDC), fully mixing and incubating to obtain the hydrogel, wherein the concentration of the polyethyleneimine solution and the carboxymethyl cellulose solution is 1-10 wt%, the mass ratio of the polyethyleneimine to the carboxymethyl cellulose is 0.5:1-5:1, and the pH is 4-8.

7. The use according to claim 5, wherein the polyethyleneimine-cellulose composite gel is prepared by Schiff base reaction, and the preparation process comprises the following steps:

mixing a polyethyleneimine solution and an oxidized cellulose solution to obtain hydrogel, wherein the concentration of the polyethyleneimine solution and the oxidized cellulose solution is 1-10 wt%, the mass ratio of the polyethyleneimine to the oxidized cellulose is 0.5:1-5:1, and the pH is 6-10.