US-12616195-B2 - Conjugates between a biopolymer and a photoactivated antimicrobial agent for combating fungal diseases of agricultural interest and methods for producing said conjugates

Abstract

This invention relates to the field of control of different fungal diseases in the agricultural industry. Specifically, the invention reports conjugates between biopolymers and a photoactivable agent, which when activated by light generates singlet oxygen, a species known for its antimicrobial capacity. The invention also relates to methods of production of these conjugates and their uses.

Inventors

• Denis Alberto FUENTEALBA PATINO
• Luciano Francesco DIBONA VILLANUEVA
• Hector Antonio VALDES GOMEZ

Assignees

• PONTIFICIA UNIVERSIDAD CATOLICA DE CHILE

Dates

Publication Date

20260505

Application Date

20201116

Priority Date

20191212

Claims (17)

1. 1 . A fungicidal composition for inhibiting growth or combatting fungal infections of fungi of the genera Botrytis, Penicillium , and Rhizopus , the fungicidal composition comprising a biopolymer selected from the group consisting of chitosan, poly-lysine, alginate, and cellulose, conjugated to a photoactivable agent selected from the group consisting of riboflavin, porphyrins, phthalocyanins, naphthalocyanins, chlorines, phenothiazines, acridines, and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY), wherein the photoactivable agent generates oxygen radicals when activated by natural light.
2. 2 . The fungicidal composition of claim 1 , wherein the biopolymer is at a concentration between 0.1% and 5% w/v, based on the total volume of the fungicidal composition.
3. 3 . The fungicidal composition of claim 1 , wherein the biopolymer is at a concentration between 0.1% and 2.5% w/v, based on the total volume of the fungicidal composition.
4. 4 . The fungicidal composition of claim 1 , wherein the photoactivable agent is at a concentration between 0.1% and 15% (w/w), based on the total weight of the biopolymer and the photoactivable agent.
5. 5 . The fungicidal composition of claim 4 , wherein the photoactivable agent is at a concentration between 0.1% and 1% (w/w), based on the total weight of the biopolymer and the photoactivable agent.
6. 6 . The fungicidal composition of claim 5 , wherein the biopolymer is at a concentration of 1% w/v based on the total volume of the fungicidal composition, and the photoactivable agent is at a concentration of 1% w/w, based on the total weight of the biopolymer and the photoactivable agent.
7. 7 . The fungicidal composition of claim 1 , wherein the biopolymer is chitosan and the photoactivable agent is riboflavin or protoporphyrin IX.
8. 8 . The fungicidal composition of claim 1 , wherein the biopolymer is chitosan and the photoactivable agent is riboflavin.
9. 9 . A method for preparing the fungicidal composition of claim 1 comprising: a) combining the photoactivable agent (FA) with 4-maleimidophenyl isocyanate (PMPI) to obtain an FA-PMPI derivative; b) treating the biopolymer (BP) with thioglycolic acid, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide to obtain a biopolymer product functionalized with reactive thiol groups (BP-SH); c) reacting the FA-PMPI derivative and the BP-SH in an aqueous solution to obtain a conjugate product; and d) optionally, dialyzing and lyophilizing the conjugate product for storage.
10. 10 . The method of claim 9 , wherein the biopolymer is chitosan and the photoactivable agent is riboflavin or protoporphyrin IX.
11. 11 . The method of claim 9 , wherein, in step a), the FA is combined with the PMPI at 45° C. in nitrogen atmosphere for 24 h to obtain the FA-PMPI derivative.
12. 12 . The method of claim 9 , wherein, in step c), the FA-PMPI derivative and the BP-SH are reacted in the aqueous solution at pH 6 for 24 hours to obtain the conjugate product.
13. 13 . The method of claim 9 , wherein the biopolymer is chitosan and the photoactivable agent is riboflavin.
14. 14 . A method of treating or controlling gray rot, green rot, or soft rot fungal infections of a plurality of fruits or vegetables comprising contacting the fruits or vegetables with the fungicidal composition of claim 1 .
15. 15 . The fungicidal composition of claim 14 , wherein the biopolymer is chitosan and the photoactivable agent is riboflavin or protoporphyrin IX.
16. 16 . The fungicidal composition of claim 14 , wherein the biopolymer is chitosan and the photoactivable agent is riboflavin.
17. 17 . A method of treating or preventing-controlling fungal infections caused by Botrytis cinerea, Penicillium digitatum , and/or Rhizopus stoloniser of a plurality of fruits or vegetables comprising contacting the fruits or vegetables with the fungicidal composition of claim 12 .

Description

FIELD OF INVENTION The invention consists of a chemical formulation based on a biopolymer, a linker and a photoactive agent, which can be used for the biological control of different pathogens of agronomic interest. The method of application of this formulation is by means of an aerosol spray and by means of visible light irradiation the photoactive effects are activated, thus inhibiting the growth of fungi of the genera Botrytis, Penicillium and Rhizopus, by the generation of singlet oxygen. BACKGROUND Agriculture is the fundamental basis for the sustainability of food security on the planet, as it represents the main source of food and is part of the economy of developing countries. Demand for food has now increased, so there is a need to optimize agricultural production. To minimize losses in agricultural production, different biological control agents are used in order to minimize losses caused by different pathogens both pre- and post-harvest. Two approaches to controlling post-harvest diseases have been described: The use and management of beneficial microflora that already exists in fruits and vegetable surfaces, or the introduction of artificial antagonists. The state of art teaches that methods to manipulate populations of microorganisms in a beneficial manner are not widely used due mainly to the limited knowledge of how these populations of microorganisms grow in the different food matrix. At present the most commonly used method of biological control is the use of different antagonists. Within this approach the elimination or reduction of pathogenic microorganisms is typically carried out by surfactants, irradiation, exposure to solvents or exposure to agents that cause oxidative damage to biological macromolecules of microorganisms. These latter treatments include gases such as ethylene oxide and chlorine dioxide. In environments where humans are present, the use of gamma-ray or high-intensity UV radiation is undesirable, as is exposure of humans to organic solvents and harmful gases. In addition, the use of biodegradable substances that do not harm the environment and are safe for human health is one of the challenges of both agriculture and the post-harvest fruit and vegetable industry. Poli-(BGA-AGA), also known generically as chitosan (although this name covers a wider family of biopolymer with different proportions between the monomers 2-amino-2-deoxi-D-glucopyranose and 2-acetamide-2-deoxi-D-glucopyranose), is a biopolymer formed from the organic or polymeric units (polymeric). of β(1-4)-2-acetamide-2-deoxy-D-glucose and monomers of β(1-4)-2-amino-2-deoxy-D-glucose, where the first monomer predominates the second in at least a 2:1 ratio. Selective interaction of Poli-(BGA-AGA) with trace metals inhibits toxin production and microbial growth. Poli-(BGA-AGA) has antifungal and antibacterial activity that can be bactericidal or bacteriostatic, depending on the strains and the specific characteristics of Poli-(BGA-AGA). The antimicrobial activity of Poli-(BGA-AGA), is influenced by the nature and/or the physico-chemical structure of each polymer. Thus, the degree of deacetylation (GD) and the length of the molecule are related to the intensity of the antifungal action. In general, the higher Gd, the greater the antimicrobial capacity. In addition, the antimicrobial activity of Poli-(BGA-AGA) depends on some factors inherent to the substrate on which it acts, such as environmental conditions (temperature and humidity), nutrient composition, pH and water activity. The use of Poli-(BGA-AGA) has reached a progressive and sustained interest in recent years, since its use prevents the accumulation of slowly degrading waste such as chitin. In addition, since Poli-(BGA-AGA) has antimicrobial properties, its application constitutes an alternative to synthetic chemicals for the conservation of edible fruits and vegetables. On the other hand, the state of the technique teaches that certain compounds such as dyes, porphyrins, fluorescenes, phenothiaziniums and phthalocyanins generate high-energy singlet oxygen, a potent antimicrobial agent, when exposed to light and air. These materials are often referred to as “light-activated antimicrobial materials” (LAAM). However, in order for these LAAM compounds to exercise their antimicrobial activity, they have to be activated very close to the microorganisms against which they are desired to act. These are inefficient if there is not an appropriate method of addressing these agents to the cell walls of pathogenic microorganisms. It has been described that one of the main problems suffered by fruit exporters is the scrap losses on arrival in the country of destination, and in the case of grapes it has been estimated that the percentage of loss reaches 15 percent. The main agent causing rejection in the exported grapes corresponds to the development of gray rot, caused by the fungus Botrytis cinerea. This fungus not only causes problems in grape production but al