Fungicidal bioemulsions

Description

1 / 14 FUNGICIDAL BIOEMULSIONS Field of Invention

[001] Rhamnolipids are surfactants of microbial origin produced mainly by the bacterium Pseudomonas aeruginosa. In addition to having the advantage of being produced from relatively low-cost sources, they exhibit high biodegradability, low toxicity, and excellent surface-active properties (MAKKAR, R.; CAMEOTRA, S. An update on the use of unconventional substrates for biosurfactant production and their new applications. Applied microbiology and biotechnology, v. 58, p. 428-434, 2002.)

[002] Essential oils are volatile and aromatic secondary metabolites produced by plants. The great diversity of components and their concentrations present in essential oils end up influencing and determining various activities of these metabolites, such as antimicrobial, insecticidal, antioxidant and acaricidal properties (BOHME, Karola et al. Antibacterial, antiviral and antifungal activity of essential oils: Mechanisms and applications. In: Antimicrobial compounds: Current strategies and new alternatives. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013. p. 51-81.).

[003] Despite the various possibilities for applying essential oils with antimicrobial activity, this has limitations due to the volatility and low water solubility of its components. These limitations can be overcome by encapsulating these essential oils in an emulsion system stabilized by a biosurfactant, aiming to improve the biological properties of the bioactive compounds by increasing the contact surface, thus requiring lower concentrations of these compounds. Background of the Invention

[004] The invention BR1120200248894A2 relates to a herbicidal composition comprising at least one essential oil as an active compound, at least one Petition 870250073900, dated 08 / 21 / 2025, page 4 / 18 2 / 14 surfactant and at least one oily substance, said composition being in the form of an emulsion.

[005] Patent BR1020210213043A2 refers to a fumigant insecticide formulation from the process of obtaining the essential oil of Myrciaria floribunda and the essential oil of Pothomorphe umbellata for the control of adult Sitophilus zeamays in corn grains, having as major constituents: (E)-Caryophyllene (26.81%) and Viridiflorol (6.64%) for Myrciaria floribunda and β-Pinene (27.33%) and α-Pinene (14.55%) for Pothomorphe umbellata. In the fumigant bioassay, a stock solution of the emulsion (tween 80) of 120 ppm was used, which was subsequently diluted to the desired concentrations. The results obtained showed that the formulation caused a reduction in the number of adult Sitophilus zeamays.

[006] Patent BR1020170254895A2 refers to the combined use of essential oils of Origanum vulgare L. (oregano) and Rosmarinus officinalis L. (rosemary) in synergistic concentrations as vegetable sanitizers, presenting itself as an alternative to the use of chemical sanitizers, aiming at the control of deteriorating and pathogenic microorganisms in these products. The emulsion of the essential oils of O. vulgare L. and R. officinalis L. was obtained by dissolving the essential oils (1.25 - 20 pL / mL) with the addition of 1% Tween 80, followed by application of the emulsion to vegetables by immersion, and subsequent rinsing in potable water for 30 seconds.

[007] The invention BR1020140134557A2 refers to a fungicidal formulation, based on essential oil and / or any other active compound of Brazilian peppertree (Schinus terebinthifolius), for application to superficial tissues of fruits, bark, leaves, stems and / or roots, in which it is desired to inactivate the mycelial or spore growth, propagation and dissemination structures, of the phytopathogen Lasiodiplodia theobromae. The formulations of the invention are presented as a solution, emulsion, lotion, powder, suspension, aerosol, spray, pour-on, gel, protective film or any pharmacologically acceptable composition, containing between 0.5% and 3% of Schinus terebinthifolius essential oil (v / v) and an emulsifier representing 10% to 30%. Petition 870250073900, dated 08 / 21 / 2025, page 5 / 18 3 / 14

[008] The invention BR1020130181870A2 refers to a fungicidal composition, based on essential oil and / or any other active principle of Lippia alba, for application to superficial tissues of fruits, peels, leaves, stems and / or roots, in which it is desired to reduce or eliminate the phytopathogen Lasiodiplodia theobromae. The formulations of the invention are presented as a solution, emulsion, lotion, powder, suspension, aerosol, spray, pour-on, gel, protective film or any pharmacologically acceptable composition, containing between 0.01% and 10% of Lippia alba essential oil (v / v), an emulsifier representing 10% to 30%, a solvent with a concentration between 10% and 30% and water.

[009] Patent PI0603005-0A2 proposes the creation of a natural fungicide containing 5% Mentha piperita essential oil (v / v) and 5% Eucalyptus sp. (v / v), in such a way that a water-in-oil emulsion is formed for application to crops by some suitable means, for example, spraying, whereby 10% of a natural emulsifier (v / v) is used for application.

[010] Invention BR1020170217639A2 relates to the use of essential oil of Origanum vulgare L. and carvacrol as stainless steel surface disinfectants, aiming at disinfecting surfaces that come into contact with food during processing. The emulsion of O. vulgare L. essential oil and carvacrol was obtained by dissolving the essential oils (1.25 - 20 pL / mL) with the addition of 1% Tween 80, followed by applying the emulsion to the surfaces for 15 minutes and then rinsing with potable water for 30 seconds. This invention has application in the area of ​​food hygiene and safety.

[011] The invention CN108272661A relates to a method for preparing a microemulsion of eucommia seed oil. The microemulsion is formed by a phase inversion emulsification method, where eucommia seed oil extracted by an aqueous enzymatic method containing a biosurfactant as the main raw material, and a cosurfactant, emulsion and water as auxiliary materials, was used.

[012] Patent BR1120170249405A2 proposes animal treatment compositions of essential oil, methods for producing and using such products and compositions. The compositions comprise an essential oil fraction including essential oil of Petition 870250073900, dated 08 / 21 / 2025, p. 6 / 18 4 / 14 oregano, thyme essential oil and cinnamon essential oil, which may include one or more emulsifiers and be in emulsion form. One of the emulsifiers may include arabinogalactan.

[013] The invention BR1020160295262A2 refers to a fungicidal formulation, based on essential oil and / or any other active compound of Lippia gracilis, for application to surface tissues of fruits, peels, leaves, flowers, stems and / or roots, in which it is desired to inactivate the mycelial or spore growth, propagation and dissemination structures, of the phytopathogens Thielaviopsis paradoxa and Colletotrichum acutatum. The formulations of the invention are presented in the form of solution, emulsion, lotion, powder, suspension, aerosol, spray, pour-on, gel, protective film or any pharmacologically acceptable composition, containing between 0.01% and 90.00% of Lippia gracilis essential oil (v / v) and an emulsifier representing 0.01% to 90.00%.

[014] The invention BR1120200248894A2 relates to a herbicidal composition comprising at least one essential oil as an active compound, at least one surfactant and at least one oily substance, said composition being in the form of an emulsion.

[015] Patent PI06030041A2 proposes a product based on 10% (v / v) Eucalyptus sp. essential oil emulsified with 10% natural emulsifier (rice lecithin or gum) (v / v) so that a water-in-oil emulsion is formed, intended for application in plantations.

[016] Patent BR1020190112174A2 refers to a technique for obtaining oil-in-water (O / W) emulsions using oils (copaiba, cumaru, and andiroba) and butters (cupuaçu and tucumã) from the Amazon region. The process is characterized by pouring the oily phase, composed of an oil or a blend of oils, a butter, and a lipophilic surfactant (Span 80), onto the aqueous phase composed of deionized water and a hydrophilic surfactant (Tween 20). Both phases are preheated, and after the emulsification process with high-performance equipment, the emulsion formed is cooled to room temperature to obtain the oil particles, encapsulated by the butter. Petition 870250073900, dated 08 / 21 / 2025, page 7 / 18 5 / 14

[017] The invention BR1020240008774A2 refers to a colloidal formulation called SNEDDS-FT-AO. Where a tannin-rich fraction (FT) was obtained from the hydroalcoholic extract of Anacardium occidentale L, it was encapsulated in a SNEDDS (self-nanoemulsion drug delivery system) type colloidal system prepared with Tween 80 and a food-grade vegetable oil. Its therapeutic efficacy was evaluated in in vitro (antioxidant and cytotoxicity) and in vivo (pulmonary anti-inflammatory) experimental models. The blank SNEDDS system (free of the FT fraction) satisfactorily solubilized the FT sample at low concentrations (SNEDDS-FT-AO 2.5 mg mL-1 and 5.0 mg mL-1). In vitro experiments showed significant results observed in tests of ascorbic acid-equivalent reducing activity, copper and iron ion chelating activity, as well as hydroxyl radical scavenging. SNEDDS-FT-AO did not exhibit cytotoxicity to healthy RAW, Vero, and L-929 cell lines.The in vivo pharmacological study in an experimental model of pulmonary inflammation via oral administration of the SNEDDS-FT-AO formulation and subsequent histopathological evaluation showed a satisfactory anti-inflammatory result.

[018] Patent BR102022006325A2 proposes to provide sustainable biolarvicidal and biofertilizing formulations comprising biosurfactants and essential oils as main active components, which have shown a synergistic effect at appropriate dosages. The formulations are initially developed by obtaining the biosurfactant, produced by fermentation from yeasts and a culture medium based on hemicellulosic hydrolysate of sugarcane bagasse. To the lyophilized cell-free fermented medium, orange essential oil is added, obtained by extraction from orange peel in a Soxhlet extractor (low-pressure extraction), using dichloromethane as the extraction solvent. After obtaining the formulations, they are characterized to determine the chemical composition and physicochemical properties, in addition to biological tests of larvicidal activity in A. aegypti and biological tests to evaluate phytotoxicity and fertilizing potential.

[019] The invention PI0901501-9A2 comprises antimicrobial compositions based on a combination of vegetable essential oils with enhanced antimicrobial efficacy, prepared by combining at least two vegetable essential oils, one Petition 870250073900, dated 08 / 21 / 2025, page 8 / 18 6 / 14 small, but effective, quantity of antimicrobial activity enhancer selected from the group comprising organic polyionic enhancers and inorganic polyionic enhancers. A preferred composition is a blend of vegetable essential oils in which at least one of the oils is oregano oil.

[020] Invention BR1020160203287B1 relates to a microparticle comprising wall material, non-ionic surfactant, essential oil of Ocimum gratissimum (basil) and essential oil of Thymus vulgaris (thyme). The invention has application in the food, pharmaceutical and cosmetic industries, as a means to increase the shelf life of the product and for antioxidant protection, either in product storage preventing its oxidation or as an additive in animal feed with the aim of producing meat with less propensity to oxidation.

[021] Patent PI0606042-0A2 deals with a process for obtaining and using repellent emulsions that can be described in terms of the non-limiting combination of 1:1:2 v / v / v of Eucalyptus citriora essential oil, an aqueous solution of 20% w / v anionic biodegradable surfactant, and mineral oil, which, mixed in the described proportion and diluted in a ratio of 1 to 20 liters of water, have proven action as a repellent against the insect Bemisia tabaci. The constituents of the essential oils are packaged in nano or supramolecular microstructures known as micelles. The surfactants present in the composition are also responsible for reducing the liquid-vegetable interfacial tension, favoring the wetting of the surface by the composition, and the described composition shows greater efficiency when administered through a pumping spray process.The anionic surfactant used is found in detergents and / or cosmetics, exemplified by sodium dodecylbenzenesulfonate, which is highly biodegradable. The mineral oil used is a mixture of non-volatile hydrocarbons and co-surfactants, which exhibit high tolerability, such as those used to treat constipation in humans. Finally, the mineral oil used acts as an emulsifying aid and helps fix the active ingredient to the surface of the plant material, since its hydrophobic characteristics confer high affinity with the surface of plants, resulting in a synergistic effect of the mineral oil. Petition 870250073900, dated 08 / 21 / 2025, p. 9 / 18 7 / 14 with the surfactant, in the fixation of eucalyptus oil to the vegetables, resulting in a more efficient formulation.

[022] Invention BR1020220172960A2 describes an antimicrobial product produced from essential oils of Cymbopogon flexuosus, Origanum vulgare, and Thymus vulgaris, among others, with proven antimicrobial activity after performing the disc diffusion method and determining the minimum inhibitory concentration (MIC) by the broth microdilution technique against Staphylococcus aureus. The essential oils can be used in their free form and combined, stabilized in the form of a cream and / or ointment. The cream was produced through an agitation process comprising water, a viscosity agent, and essential oils as active agents. This cream can be used in the treatment and / or prevention of infections caused by S. aureus, such as bovine mastitis.

[023] Patent BR112021010369-4A2 describes an aqueous nanoemulsion formulation comprising an oil, a solvent, a sorbate, and a saponin in sufficient quantity to form a nanoemulsion of said oil in water and sufficient water to make up 100 percent by weight. The report also describes the use of the nanoemulsion formulation as a disinfectant, pesticide, and / or plant growth regulator.

[024] The patents found describe: 1) a herbicidal composition in emulsion form composed of an essential oil, at least one surfactant and an oily substance; 2) a fumigant insecticidal formulation in emulsion form composed of essential oils of Myrciaria floribunda and Pothomorphe umbellata and Tween 80; 3) plant sanitizers composed of essential oils of Origanum vulgare L. (oregano) and Rosmarinus officinalis L. (rosemary) combined with 1% Tween 80, forming an emulsion; 4) a fungicidal formulation based on essential oil of Schinus terebinthfolius (Brazilian peppertree) and an emulsifier; 5) a fungicidal composition based on essential oil of Lippia alba, emulsifier and solvent; 6) a natural fungicide based on essential oil of Mentha piperita and Eucalyptus sp. and natural emulsifier forming a water-in-oil type emulsion; 7) disinfectant product for stainless steel surfaces based on essential oil of Origanum vulgare L. and carvacrol with added Tween 80 at 1% forming a Petition 870250073900, dated 08 / 21 / 2025, p. 10 / 18 8 / 14 emulsion; 8) microemulsion formed from eucommia seed oil and a biosurfactant; 9) emulsion for animal treatment containing essential oils of oregano, thyme and cinnamon and emulsifier; 10) fungicidal formulation based on essential oil of Lippa gracilis (rosemary) and an emulsifier; 11) herbicidal composition in emulsion form based on essential oil, surfactant and at least one oily substance; 12) natural fungicide in emulsion form based on essential oil of Eucalyptus sp emulsified with rice lecithin or gum; 13) cosmetic applications from obtaining oil-in-water emulsions based on oils and butters, lipophilic surfactant (Span 80) and a hydrophilic surfactant (Tween 20); 14) Therapeutic applications against COVID-19 of a colloidal system containing a mixture of tannins from A. occidentale L.14) Tween 80 and food-grade vegetable oil; 15) biolarvicidal and biofertilizing formulations containing biosurfactants of fungal origin and essential oils as synergistic active components; 16) combination of vegetable essential oils with added organic and / or inorganic poly-ionic antimicrobial activity enhancers; 17) antioxidant microparticles of non-ionic surfactants, essential oil of basil (Ocimum gratissimum) and thyme (Thymus vulgaris); 18) repellent emulsions composed of Eucalyptus citriora essential oil, aqueous solution of biodegradable anionic surfactant and mineral oil; 19) antimicrobial cream with essential oils of Cymbopogon flexuosus, Origanum vulgare and Thymus vulgaris; 20) aqueous nanoemulsion composed of an oil, a solvent, a sorbate and a saponin.

[025] In turn, the present invention relates to the development of a biofungicide from emulsions of essential oils and rhamnolipids with antifungal activity. For the emulsions, essential oils of Cymbopogon flexuosus (Lemongrass), Cymbopogon winterianus (Citronella) and Melaleuca alternifolia were used, as well as citral (major component of Cymbopogon flexuosus oil). Each essential oil was mixed with a rhamnolipid solution from the Pseudomonas aeruginosa PA01 strain. Antifungal activity tests were performed in 96-well plates to determine the Minimum Inhibitory Concentration (MIC) and in Petri dishes to determine the Minimum Fungicidal Concentration (MFC). The Checkerboard test was performed to verify the activity between the compounds. From the results obtained we can Petition 870250073900, dated 08 / 21 / 2025, page 11 / 18 9 / 14 observe that the presence of rhamnolipids in the emulsion was able to reduce the MIC concentration obtained using only the essential oil, while the checkerboard showed a synergistic effect between the essential oils and rhamnolipids. Summary of the Invention

[026] The present invention relates to the use of emulsions of essential oils and rhamnolipids for the development of biofungicides. The emulsions promote increases in fungistatic and fungicidal effects of up to 66 times, as well as synergistic effects in the interaction between the compounds, when compared to essential oils and rhamnolipids used separately. The emulsions allow the application of the oils in aqueous solutions, expanding the application potential of these fungicides of natural origin. Thus, the present invention relates to an ecologically viable alternative of emulsions of natural compounds (essential oils and rhamnolipids) with synergistic antifungal effects. Detailed Description of the Invention

[027] In this process, essential oils (EOs) of Lemongrass (Cymbopogon flexuosus), Citronella (Cymbopogon winterianus) and Melaleuca alternifolia, acquired from the company FERQUIMA Indústria e Comércio Ltda, São Paulo - Brazil, and rhamnolipids (RL) produced by submerged fermentation of the Pseudomonas aeruginosa PA01 strain were used to prepare the emulsions. The emulsions were prepared in 1:1, 1:2 and 2:1 ratios starting from an initial concentration of 1-3% (w / v). The mixtures were vortexed for 30 to 90 seconds to form emulsions of essential oils stabilized by rhamnolipids.

[028] The assays for determining the Minimum Inhibitory Concentration (MIC) were performed in triplicate by broth microdilution test in 96-well plates against strains of filamentous fungi (Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Fusarium oxysporum and Fusarium verticilioides) and yeast-like fungi (Candida albicans ATCC 90028), according to CLSI document M38-A2 (CLSI. M38-A2 Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi Approved Standard—Second Edition. 28 2008. p. 13.), with adaptations for filamentous fungi. Petition 870250073900, dated 08 / 21 / 2025, page 12 / 18 10 / 14 and CLSI document M27-A2 (Clinical and Laboratory Standards Institute (CLSI). Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved Standards M27-A2. Wayne, Pa.: National Committee for Clinical Laboratory Standards: 2008), for yeasts. As a negative control, RPM I 1640 medium (with glutamine and phenol red, without bicarbonate - Gibco™, ThermoFisher Scientific) supplemented with MOPS buffer (INLAB, São Paulo, BR) (pH previously adjusted to 7.2 - 7.4 and vacuum filtered) was tested separately and in combination with essential oils (8% v / v), rhamnolipids (20% w / v), emulsions (2% w / v), without the addition of inoculum. For fungal growth control (positive control), the strains were inoculated in RPMI 1640 medium adjusted to 10⁴ CFU / mL for filamentous fungi and 10³ CFU / mL for yeasts.The MIC (Figure 1) was defined as the lowest concentration with no visible fungal growth after 48h (yeasts) and 72h (filamentous fungi) of incubation at 37°C. At the concentrations with no visible fungal growth, 10 pL of the contents of the respective wells were plated onto Sabouraud agar to obtain the Minimum Fungicidal Concentration (MFC). The MFC was defined as the lowest concentration of antifungal agents at which up to 3 colonies grew within 24h at 37°C. The tests were performed in triplicate.

[029] The interaction of the antifungal agents was also evaluated (Checkerboard) (Figure 2) following the methodology of Bocate et al. (BOCATE, Karla Paiva et al. Antifungal activity of silver nanoparticles and simvastatin against toxigenic species of Aspergillus. International journal of food microbiology, v. 291, p. 79-86, 2019.), with adaptations. The plates were incubated for 72h at 37°C for filamentous fungi and 48h at 37°C for yeast; after the incubation period, a visual reading was performed. To estimate the interaction of the compounds, the Fractional Inhibitory Concentration Index (FICI) was calculated. ICIF = MICa in combination MICb in combination isolated MIC Isolated MICb

[030] Petition 870250073900, dated 08 / 21 / 2025, page 13 / 18 11 / 14

[031] Where ICIF < 0.5 indicates synergism between the compounds, 0.5 < ICIF < 1 indicates adism, 1 < ICIF < 4 indicates indifference and ICIF > 4 indicates antagonism (SILVA, Ricardo Augusto Alves. Analysis of Growth Inhibition of Dermatophyte Fungi by Biogenic Silver Nanoparticles, Azole Compounds and Simvastatin. 2019. 59 pp. Dissertation (Master's in Microbiology) - State University of Londrina, Londrina. 2019.).

[032] Table 1 shows the fractional inhibitory concentration (FIC) in the interaction between Rhamnolipids - Lemongrass, Rhamnolipids - Citronella and Rhamnolipids - Melaleuca alternifolia. The results demonstrate that the interaction between Rhamnolipids - Lemongrass showed a synergistic effect between the components for the microorganisms Aspergillus flavus, Aspergillus niger and Candida albicans, evidenced by the reduction in the percentage of MIC values ​​of Rhamnolipids and Lemongrass, while for Fusarium verticillioides, the FIC value indicates that the interaction between these compounds is indifferent. For the rhamnolipid-citronella interaction tests, assays with Aspergillus niger, Candida albicans, and Fusarium verticillioides showed a synergistic effect, exhibiting a percentage reduction in the MIC values ​​of RL and CT, while for Aspergillus flavus, the observed interaction was one of indifference. For the rhamnolipid-Melaleuca alternifolia interaction test, the microorganisms A.For A. flavus, A. niger, and C. albicans, the compounds showed a synergistic effect with each other, presenting a reduction in the MIC values ​​of RL and the essential oil of ML, while for Fusarium verticillioides the interaction found was aditism.

[033] Table 1. Fractional Inhibitory Concentration Index (FICI) of essential oils and rhamnolipid against filamentous fungi and yeast species. Species Association Individual MIC (%) Association MIC (%) ICIF % Red MIC Aspergillus flavus RL - CL 5 / 1 0.039 / 0.25 0.257 99.22 / 75 RL - CT 5 / 1 0.039 / 2 2.007 99.22 / - RL - ML 5 / 2 0.039 / 0.5 0.257 99.22 / 75 Aspergillus niger RL - CL 2.5 / 0.25 0.039 / 0.015 0.075 98.44 / 94 Petition 870250073900, dated 08 / 21 / 2025, page 14 / 18 12 / 14 RL - CT 2.5 / 1 0.039 / 0.015 0.03 98.44 / 98.5 RL - ML 2.5 / 1 0.039 / 0.06 0.075 98.44 / 94 Candida albicans RL - CL 5 / 0.5 0.039 / 0.031 0.069 99.22 / 93.8 RL - CT 5 / 1 0.039 / 0.06 0.067 99.22 / 94 RL - ML 5 / 2 0.039 / 0.25 0.132 99.22 / 87.5 Fusarium verticillioides RL - CL 5 / 0.5 0.039 / 0.5 1.007 99.22 / - RL - CT 5 / 2 0.039 / 0.5 0.257 99.22 / 75 RL - ML 5 / 1 0.039 / 0.5 0.507 99.22 / 50 RL: rhamnolipid; CL: lemongrass essential oil; CT: citronella essential oil; ML: tea tree essential oil; ICIF: Fractional Inhibitory Concentration Index; % Red MIC: Percentage Reduction of Minimum Inhibitory Concentration = (individual MIC - combination MIC) x 100 / individual MIC.

[034] Tables 2 and 3 show, respectively, the minimum inhibitory concentrations and minimum fungicidal concentrations of RL solutions, EO solutions, and emulsions of essential oils and rhamnolipids. The results obtained demonstrate that all fungi tested showed sensitivity to all antifungal agents. However, when essential oils are stabilized in rhamnolipid emulsions, the minimum inhibitory and fungicidal concentrations can be up to 66 times lower, depending on the fungus, when compared to the MICs obtained using only the essential oil.

[035] Table 2. Minimum inhibitory concentration (MIC) (%) of essential oils, rhamnolipids and emulsions of essential oils and rhamnolipids against filamentous fungal and yeast species. Antifungal agents Aspergillus flavus Aspergillus fumigatus Aspergillus niger Candida albicans Fusarium oxysporum Fusarium verticillioides CL 1 0.25 0.25 0.5 1 0.5 CT 1 1 1 1 0.25 2 ML 2 2 1 2 0.5 1 RL 5 > 5 2.5 5 > 5 5 RL + CLa 0.015 0.031 0.015 0.015 0.015 0.015 RL + CTa 0.031 0.125 0.015 0.031 0.031 0.031 RL + MLa 0.25 0.25 0.062 0.125 0.125 0.125 Petition 870250073900, dated 08 / 21 / 2025, p. 15 / 18 13 / 14 CL: Lemongrass essential oil; CT: Citronella essential oil; ML: Melaleuca alternifolia essential oil; RL: rhamnolipid; RL + CL: emulsion of lemongrass essential oil stabilized by rhamnolipid; RL + CT: emulsion of citronella essential oil stabilized by rhamnolipid; RL + ML: emulsion of Melaleuca alternifolia essential oil stabilized by rhamnolipid. RL:EO mixture (1:1)

[036] Table 3. Minimum Fungicidal Concentration (MFC) (%) of essential oils, rhamnolipids and emulsions of essential oils and rhamnolipids against filamentous fungi and yeast species. Antifungal agents Aspergillus flavus Aspergillus fumigatus Aspergillus niger Candida albicans Fusarium oxysporum Fusarium verticillioides CL 1 0.25 0.25 0.5 1 0.5 CT 1 1 1 1 0.25 2 ML 2 2 1 2 0.5 1 RL 5 > 5 2.5 5 > 5 5 RL + CLa 0.015 0.031 0.015 0.015 0.015 0.015 RL + CTa 0.031 0.125 0.015 0.031 0.031 0.031 RL + MLa 0.25 0.25 0.062 0.125 0.125 0.125 CL: Lemongrass essential oil; CT: Citronella essential oil; ML: Melaleuca alternifolia essential oil; RL: rhamnolipid; RL + CL: emulsion of lemongrass essential oil stabilized by rhamnolipid; RL + CT: emulsion of citronella essential oil stabilized by rhamnolipid; RL + ML: emulsion of Melaleuca alternifolia essential oil stabilized by rhamnolipids. RL:EO mixture (1:1)

[037] Table 4 shows the minimum inhibitory and minimum fungicidal concentrations of RL solutions, Lemongrass EO, Citral and rhamnolipid emulsions. As can be seen, both Lemongrass essential oil and Citral, the major component in the composition of this oil, exhibit antifungal activity alone or in combination with RL, with the combinations having synergistic activity.

[038] Table 4. Minimum inhibitory concentration (MIC) (%) and Minimum fungicidal concentration (MFC) (%) of essential oils, rhamnolipid and rhamnolipid-stabilized essential oil emulsions against filamentous fungal and yeast species. Antifungal agent Aspergillus flavus Aspergillus niger Candida albicans Fusarium verticillioides MIC MFC MIC MFC MIC MFC MIC MFC CL 1 1 0.25 0.25 0.5 0.5 0.5 0.5 Citral 2 2 0.125 0.125 1 1 1 1 Petition 870250073900, dated 08 / 21 / 2025, page 16 / 18 14 / 14 RL 5 5 2.5 2.5 5 5 5 5 RL + CL at 0.015 0.015 0.015 0.015 0.015 0.015 0.015 0.015 RL+ Citral at 0.031 0.031 0.047 0.047 0.031 0.031 0.031 0.031 CL: Lemongrass essential oil; RL: rhamnolipid; RL + CL: emulsion of lemongrass essential oil stabilized by rhamnolipid; RL + Citral: emulsion of citral stabilized by rhamnolipids; RL:EO mixture (1:1) Figure Captions

[039] Figure 1 Representation of the dilutions (1) of antifungal agents for determination of the Minimum Inhibitory Concentration (MIC). (A) Essential oil dilutions - EO (2); (B) Rhamnolipid dilutions - RL (3); (C) Essential oil emulsion dilutions stabilized by rhamnolipids - RL + EO (4); C+: positive control (absence of antifungal agent); C-: negative control (absence of antifungal agent and microorganism).

[040] Figure 2 Representation of the assay for evaluating the interaction between antifungal agents (Checkerboard); Compound A (1); Dilution Compound A (2); Compound B (3); Dilution Compound B (4). Petition 870250073900, dated 08 / 21 / 2025, page 17 / 18

Claims

1 / 1 CLAIMS 1. FUNGICIDAL BIOEMULSIONS, characterized by emulsions of essential oils and / or their fractions and rhamnolipids with synergistic antifungal activities; the essential oils from Cymbopogon flexuosus (Lemongrass), Cymbopogon winterianus (Citronella), Melaleuca alternifolia and the rhamnolipids produced by the bacterium Pseudomonas aeruginosa by submerged cultivation, the mass ratio of essential oil and rhamnolipids varying from 1:1 to 1:2 m / v; and water varying from 1:400 to 1:7000.

2. FUNGICIDAL BIOEMULSIONS, according to claim 1, characterized by antifungal activity against phytopathogenic and mycotoxigenic fungi of agronomic importance.

3. FUNGICIDAL BIOEMULSIONS, according to claim 1, characterized by antifungal activity against yeasts and filamentous fungi of medical importance. Petition 870250007901, dated 01 / 30 / 2025, pp. 22 / 32

Images