RU2432741C1 - Fungicide material for food and agricultural products - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. The fungicide material consists of 0.01-10 wt % polyene antifungal agent in form of natamycin or lucensomicin or nystatin or amphotericin B; 0.01-10 wt % antifungal organic acid in form of benzoic acid or propionic acid or sorbic acid; 80-99.98 wt % copolymer of N-vinyl pyrrolidone and allyl glycidyl ether with molecular weight 2000-50000 consisting of N-vinyl pyrrolidone and allyl glycidyl ether links in molar ratio 1-10:1, with ester groups.

EFFECT: invention enables to prolong fungicidal activity and increases the level of antifungal activity.

4 ex, 2 tbl

Description

The invention relates to the field of biotechnology, in particular to fungicidal materials with controlled and prolonged release of an antifungal active agent for combating fungal infections and damage to food, agricultural products and plants.

In recent decades, special attention has been paid to improving the effectiveness of biologically active and medicinal substances by creating their forms with controlled and prolonged release of the active substance. One of the most promising approaches to the creation of such forms is to obtain preparations that are a biologically active substance associated with a certain carrier (for example, polymeric) biodegradable bonds (hydrolyzable fragments, ionic, hydrogen bonds). This structure provides a prolonged release of the active substance during its gradual separation from the carrier and provides increased efficiency of the entire system at lower concentrations of the active component compared to its usual forms.

This approach seems promising for the creation of new means of combating fungal infections of food and agricultural products.

Each year, 30-40% of food and fodder in the world is affected by fungal mycotoxins with a corresponding effect on human health and the economy. The most common mycotoxins are aflatoxins, ochratoxins, trichothecenes, zearalenone, which are found in a number of products, such as dry animal feed, cereals, coffee, cheese, fruits, vegetables, etc.

In recent years, two groups of fungicides have been the most effective means of combating fungal infections: polyene antifungal antibiotics (natamycin, nystatin, amphotericin, etc.) and carboxylic acids (acetic acid, propionic acid, sorbic acid and their salts).

For example, natamycin has been used successfully for more than 20 years to prevent the growth of molds on foods such as cheeses and sausages. But, despite the high activity of natamycin, its poor solubility in water and the existence of certain types of fungi that are resistant to this antibiotic, for example, Verticilium cinnabarimam, Butrytis cinerea, Trichophyton species and Penicillium echinulatum, lead to infection of some of the processed products.

The harmful effects of microflora on food and agricultural products can also be controlled and prevented using preparations of organic acids and their salts. For example, sorbic acid, propionic acid, benzoic acid and their salts stop the growth of fungi and reduce the possibility of biosynthesis of mycotoxins by inhibiting biological transformation pathways. The disadvantage of fungicidal preparations based on organic acids is that they are effective only at high concentrations, which leads to an increase in the toxicity of such drugs, the manifestation of an irritating effect on the digestive system of humans and animals, as well as to a deterioration in the taste of food products. In addition, as in the case of polyene antibiotics, there are a number of fungi that are resistant to the antifungal effect of carboxylic acids.

In connection with the aforementioned disadvantages of both polyene antifungal antibiotics and fungicidal organic acids, it seems promising to use fungicidal preparations and materials containing both of these types of antifungal objects.

To date, a fairly large number of examples are known of using combinations of polyene antifungal antibiotics together with organic acids in various proportions and in various solvents as fungicidal preparations.

So, for example, patent DE 2529532 (Gist-Brocades N.V.) describes a combination of natamycin and an organic acid in a water-alcohol solution. This combination is effective in protecting citrus fruits from mold. Due to the higher solubility of natamycin in this preparation, the growth of fungi is inhibited not only on the surface, but also in the depths of the fetus. The disadvantage of this system is the need for the use of alcohols and lower stability of natamycin compared to its aqueous preparations.

Patent J 61146153 (Nippon Nosan Kogyo) describes the use of an antibiotic and antibacterial substance in an aqueous solution of an organic acid to prevent damage to mold and bacteria of mulberry leaves. In this case, it is proposed to use a macrocyclic antibiotic (for example, natamycin) as an antibiotic, and sorbic acid, sorbates, propionic acid or propionates as an antibacterial component.

DE 848934 (Gist-Brocades N.V.) describes liquid antifungal compositions containing natamycin and an organic carboxylic acid for treating diseases of animals, in particular livestock. In this case, it is proposed to use mainly citric acid as an organic acid. Such a liquid preparation has a pH of 2-4.5. The drug is especially effective for treating cattle infections caused by the fungus Trichophyton verrucosum, which is more resistant to natamycin than mold fungi (the minimum inhibitory concentration or MIC for Trichophyton verrucosum species is 12.5-25.0 μg / ml, whereas for molds affecting food products, IPC is 1-10 μg / ml). The increased effectiveness of the composition is also associated with an increase in the solubility of natamycin at low pH values. The disadvantage of this method is that natamycin is quite unstable at low pH values, and this leads to the fact that the drug is effective for a short period of time.

The closest solution to the proposed is the fungicidal material proposed in the patent US 5597598, and which is an aqueous alcohol-free composition containing 0.005-2.0 wt.% Polyene antifungal agent, 0.01-10.0 wt.% Acid antifungal agent from the series benzoic acid, propionic acid, sorbic acid and their salts, and 0.01-10.0 wt.% additives of an acid or alkali metal salt of an acid from a radic acetic acid, lactic acid. Polyene antifungal agent is used from a number of natamycin, lucensomycin, nystatin, amphotericin B. The disadvantage of this method is the low solubility of polyene antifungal antibiotics in water and high concentrations of organic carboxylic acids in the preparation, which leads to an increase in the toxicity of the preparation and deterioration in the taste of processed products. In addition, the use of pure low molecular weight fungicidal agents leads to very short shelf life of the fungicidal composition.

In this case, the technical result consists in increasing the fungicidal action of the new material compared to pure low molecular weight fungicides due to the gradual prolonged release of active antifungal components of the system from the carrier polymer, as well as in a higher level of antifungal activity of the new material compared to the mixture of low molecular weight fungicides same concentration.

The specified technical result is achieved by the fact that the proposed fungicidal material for food and agricultural products consists of 0.01-10 wt.% Polyene antifungal agent, which is used either natamycin, or lucensomycin, or nystatin, or amphotericin B, and 0.01 -10 wt.% Antifungal organic acid, which is used as either benzoic acid, or propionic acid, or sorbic acid, and additionally contains 80-99.98 wt.% Copolymer of N-vinylpyrrolidone and allyl glycidyl ether as carrier polymer.

As the carrier polymer for the fungicidal material, a copolymer of N-vinylpyrrolidone and allyl glycidyl ether (PVP-AGE) with a molecular weight of 2000-50000, which consists of N-vinyl pyrrolidone and allyl glycidyl ether units in a molar ratio of 1-10: 1, is used. The chemical structure of the used carrier polymer is presented below

Polyene antibiotics used as a fungicidal agent form a complex with a carrier polymer due to the formation of hydrogen bonds with N-vinylpyrrolidone units.

Used as fungicidal agents, organic acids form a complex with a carrier polymer due to the formation of an ester bond with the units of the allyl glycidyl ether of the carrier polymer.

Moreover, the gradual hydrolysis of the polymer fungicidal material ensures its antifungal effect for a long time.

In addition, the proposed fungicidal material is obtained, and it can be used in the form of a 0.001-20 wt.% Aqueous solution.

The essence of the invention is illustrated by the following examples of specific implementation.

Example 1. Prepare fungicidal material from a mixture of 99.8 wt.% A copolymer of N-vinylpyrrolidone and allyl glycidyl ether with a molecular weight of 2000 (molar ratio of monomers 4: 1, respectively), 0.1 wt.% Nystatin and 0.1 wt.% Benzoic acids. The resulting mixture was dissolved in water, stirred vigorously at 50 ° C for 8 hours to obtain a polymer complex of amphotericin and sorbic acid. Separation of non-attached fungicides is carried out by centrifugation and then dialysis. The fungicidal material is freeze-dried.

Example 2. Prepare fungicidal material from a mixture of 98.0 wt.% A copolymer of N-vinylpyrrolidone and allyl glycidyl ether with a molecular weight of 50,000 (molar ratio of monomers 3: 1, respectively), 1.0 wt.% Amphotericin and 1.0 wt.% Sorbic acids. The resulting mixture was dissolved in water, stirred vigorously at 50 ° C for 8 hours to obtain a polymer complex of amphotericin and sorbic acid. Separation of non-attached fungicides is carried out by centrifugation and then dialysis. The fungicidal material is freeze-dried.

Example 3. Prepare a fungicidal material from a mixture of 80.0 wt.% A copolymer of N-vinylpyrrolidone and allyl glycidyl ether with a molecular weight of 10000 (molar ratio of monomers 2: 1, respectively), 10 wt.% Natamycin and 10 wt.% Propionic acid. The resulting mixture was dissolved in water, stirred vigorously at 50 ° C for 8 hours to obtain a polymer complex of amphotericin and sorbic acid. Separation of non-attached fungicides is carried out by centrifugation and then dialysis. The fungicidal material is freeze-dried.

Example 4. Prepare an aqueous solution with a concentration of 10 wt.% Fungicidal material from a mixture of 80.0 wt.% A copolymer of N-vinylpyrrolidone and allyl glycidyl ether with a molecular weight of 10,000 (molar ratio of monomers 3: 1, respectively), 10 wt.% Natamycin and 10 wt.% propionic acid by dissolving the components in water with vigorous stirring at 50 ° C for 8 hours.

To confirm the achievement of the technical result provided by the proposed invention, the antifungal activity of the fungicidal material obtained in Examples 1-3 was studied in comparison with pure low molecular weight fungicidal agents, as well as the duration of the fungicidal effect of the obtained material compared to low molecular weight analogues.

The antifungal activity of the fungicidal material was determined in relation to the culture of the fungus Fusarium oxysporum by the “paper disk” method. The essence of this method consists in applying to a paper disk, placed on the surface of an agar medium with a test sample of the fungus, a fungicide test solution and observing the radial growth rate of the fungus under these conditions.

Antifungal activity (AA) was evaluated by the formula

AA (%) = 100 - sample colony diameter / standard colony diameter × 100%

The experiments were carried out at 25 ° C for 7 days, pH of 6.4. Each given value is the arithmetic mean of three parallel experiments. For comparison, AA was measured in an aqueous solution of a mixture of pure antifungal antibiotic and fungicidal acid at the same concentration as in the sample of polymeric fungicidal material.

Table 1 shows data that confirm that the antifungal activity of a polymer fungicidal material containing an antifungal antibiotic and fungicidal acid is higher than the antifungal activity of a solution of a mixture of pure antifungal antibiotic and fungicidal acid in the same concentration. Therefore, to achieve a certain level of antifungal activity in the case of the proposed fungicidal material, a smaller amount of fungicidal agents is needed.

Table 1 Antifungal activity in relation to the culture of the fungus Fusarium oxysporum A drug Antifungal activity of the drug,% 99.8 wt.% PVP-AGE + 0.1 wt.% Nystatin + 0.1 wt.% Benzoic acid. 8.2% 0.1 wt.% Nystatin + 0.1 wt.% Benzoic acid. 6.4% 98.0 wt.% PVP-AGE + 1.0 wt.% Amphotericin + 1.0 wt.% Sorbic acid. 46.7% 1.0 wt.% Amphotericin + 1.0 wt.% Sorbic acid. 41.1% 80.0 wt.% PVP-AGE + 10.0 wt.% Natamycin + 10.0 wt.% Propionic acid. 76.5% 10.0 wt.% Natamycin + 10.0 wt.% Propionic acid. 64.8%

To determine the time of effective antifungal exposure of the fungicidal material, an experiment using agar medium was used. To do this, 0.15 ml of a spore suspension of Penicillium echinulatum strain fungi was placed and evenly distributed in a Petri dish with a diameter of 150 mm with an agar medium. After incubation of the medium for 12 hours at 6 ° C, a solution of fungicidal material or a solution of a mixture of pure fungicidal agents was added to the plates for comparison. After that, the samples were incubated at 15 ° C and a relative humidity of 95%. Each day, the number of visible colonies of fungi formed was calculated and their average value was calculated. After the number of colonies in the plate exceeded 50, it was believed that the sample was completely covered with mold, and the elapsed time (in days) from the beginning of the experiment was taken as the duration of the antifungal action of the material. The research results are presented in Table 2.

table 2 Comparison of the duration of the antifungal effect of various fungicidal preparations on the growth of the fungus culture of Penicillium echinulatum. A drug Duration of antifungal action, days 99.8 wt.% PVP-AGE + 0.1 wt.% Nystatin + 0.1 wt.% Benzoic acid. 10 0.1 wt.% Nystatin + 0.1 wt.% Benzoic acid. 2 98.0 wt.% PVP-AGE + 1.0 wt.% Amphotericin + 1.0 wt.% Sorbic acid. fourteen 1.0 wt.% Amphotericin + 1.0 wt.% Sorbic acid. 3 80.0 wt.% PVP-AGE + 10.0 wt.% Natamycin + 10.0 wt.% Propionic acid. 26 10.0 wt.% Natamycin + 10.0 wt.% Propionic acid. 6

As can be seen from table 2, the proposed fungicidal material based on a carrier polymer with immobilized fungicidal agents provides several times longer antifungal effect compared to low molecular weight fungicides with the same content of active components in the preparations.

Thus, the proposed fungicidal material, which is a polymer complex of a mixture of fungicidal agents of two different classes (polyene antibiotics and organic acids), provides higher antifungal activity and a longer antifungal effect compared to conventional solutions of mixtures of low molecular weight fungicides in the same concentrations.

Claims (5)

1. Fungicidal material for food and agricultural products, consisting of 0.01-10 wt.% Polyene antifungal agent, which is used either natamycin, or lucensomycin, or nystatin, or amphotericin B, and 0.01-10 wt.% antifungal organic acid, which is used as either benzoic acid, or propionic acid, or sorbic acid, characterized in that it further comprises 80-99.98 wt.% copolymer of N-vinylpyrrolidone and allyl glycidyl ether with a molecular weight of 2000-50000, in the following is correlated and, wt.%:
antifungal agent 0.01-10.0 antifungal acid 0.01-10.0 copolymer of N-vinylpyrrolidone and allyl glycidyl ether 80-99.98 2. The material according to claim 1, characterized in that the copolymer of N-vinylpyrrolidone and allyl glycidyl ether consists of units of N-vinyl pyrrolidone and allyl glycidyl ether in a molar ratio of 1-10: 1. 3. The material according to claim 2, characterized in that the N-vinylpyrrolidone units of the copolymer of N-vinylpyrrolidone and allyl glycidyl ether are bonded to the polyene antibiotic molecules by hydrogen bonds. 4. The material according to claim 2, characterized in that the units of allyl glycidyl ether of a copolymer of N-vinylpyrrolidone and allyl glycidyl ether are linked with ester bonds by molecules of antifungal organic acid. 5. The material according to claim 1, characterized in that it is used in the form of a 0.001-20 wt.% Aqueous solution.