Biological antifungal inoculant for coffee fruits.

Description

/ 10 “ANTIFUNGAL BIOLOGICAL INOCULANT FOR COFFEE FRUITS” FIELD OF THE INVENTION

[001] This invention patent relates to a biological inoculant, whose formulation contains yeasts of the genera Pichia, Hanseníaspora and Meyorozyma, which inhibits the growth of deleterious fungi in coffee fruits and thus maintains the quality and wholesomeness of the beverage. This invention is intended for the agricultural sector, specifically coffee growing. FUNDAMENTALS OF THE INVENTION

[002] The growth of filamentous fungi causes damage to coffee production. These fungi produce substances that impair the quality of the beverage: acetic acid, chlorogenic acid, pyrazines and pyrroles, and can even be harmful to human health, such as Ochratoxin A. These substances, added to the coffee fruits during fermentation, drying and storage processes, result in low beverage quality and lower market value.

[003] The control of these fungi already occurs mainly in stored grains with the use of chemical substances. However, it is common knowledge that the chemical control of agricultural pathogens also poses risks to the environment and human health.

[004] Therefore, it is necessary to develop alternative controls for agricultural pathogens; among the control methodologies, we highlight biological control here.

[005] The following documents are presented in the state of the art that aim to present biological control solutions for the technical problem caused by crop pathogens. Petition 870250050109, dated 06 / 16 / 2025, page 5 / 16 2 / 10

[006] Patent CR 20190231 A requests protection for a biological liquid substance with antifungal action; however, it does not mention its application to coffee fruits and does not contain yeasts of the genera mentioned above in its composition. The document refers to a liquid biological product with antifungal action to be applied to soils in order to control the presence of pathogenic fungi of the genera Botrytis, Fusarium, and Phytophthora. The product's composition has as its main component oospores of Pythium oligantrum, a parasite that attacks and causes the death of fungi. The applied composition contains a saline solution, biomass of mycoparasite oospores comprising between 0.05% and 0.10%, stabilized in sucrose between 60.0% and 64.5%, diluted in 90% to 95% water. The number of oospores per ml should be between 1x10³ and 2x10⁷.Pythium oligantrum osmolytes must be stabilized in an oil, which may be paraffin oil, mineral oil, glycerol, or sunflower oil, at a concentration of 79.77% to 99.9% by weight. The parasite is cultivated in a solid medium containing millet grains and husks as an energy source. The product in question is an antifungal effective against soil-borne fungi; however, the application does not specify its use in coffee fruits.

[007] Patent WO 2021 / 050927 A2 uses yeasts of the genus Meyorozyma to inhibit the growth of filamentous fungi, but does not request protection of this activity in coffee fruits. The document in question is a request for protection of a biological composition of yeast strains with antifungal action to be applied to parts of vegetables that can be used as food. The application includes a series of yeast species that can be used in the composition. The product to be applied is an exudate from the composition of one or more yeasts cultivated in a culture medium containing crushed food waste. The supernatant of this exudate must contain in its composition an organic acid, an enzyme, a branched alcohol, a cyclopeptide, an aldehyde, or a ketone. The applied product Petition 870250050109, dated 06 / 16 / 2025, page 6 / 16 3 / 10 acts in an antagonistic system against pathogenic fungi of various species, however, there is no description for protection against fungi of the genus Aspergillus, which is one of the main pathogens in coffee beans and fruits and one of the main fungi affected by our antifungal composition.

[008] Patent CN 115161224 A refers to a biological product for the production and improvement of coffee beverage quality, but makes no mention of combating fungi nor does it use yeasts of the genera Pichia, Hanseníaspora, and Meyorozyma. The document in question is a patent application for a biological product based on microorganisms of the genus Bacillus, for application in coffee fruits subjected to the fermentation process. The microorganisms used in the composition were isolated from coffee fruits and have the ability to induce the synthesis of chlorogenic acid, to be used in the fermentation process. Characteristics of the composition, method of application, culture medium, and formulation of the product are not available. Despite its application in coffee cultivation, the use of yeasts in the product's composition is not reported. Furthermore, the patent application does not specify its target of action, which in our case are pathogenic fungi that are detrimental to the quality of the coffee beverage.

[009] Article 1 (https: / / doi.Org / 10.1016 / j.ijfoodmicro.2005.06.015) presents scientific evidence that yeasts of the genera Pichia and Hanseníaspora are capable of inhibiting the growth of fungi of the genus Aspergillus ochraceus and the production of ochratoxin A (OTA) in culture media containing malt extract agar and coffee agar. However, there are no reports in this article about experiments under field conditions.

[0010] 0 Article 2 (https: / / doi.Org / 10.1016 / j.foodres.2018.10.002) refers to an antifungal activity of the yeasts Saccharomyces cerevisiae and Candida ethanolica during the fermentation of cocoa beans, however it does not report the genera Pichia, Hanseníaspora and Meyorozyma and does not report the efficiency of this activity in coffee fruits. Petition 870250050109, dated 06 / 16 / 2025, page 7 / 16 / 10

[0011] The present invention was developed in view of the above circumstances and aims to provide a biological product to coffee growers, so that they can apply it to their crops in order to inhibit the growth of deleterious fungi that naturally grow on the fruits during the drying process and, thus, improve the quality of the fruit and the coffee beverage. The present invention does not contain toxic elements in its composition, does not harm the environment, adds quality gain to the fruits and the coffee beverage, representing an increase in market value for commercialization and protection of consumer health. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 presents an in vitro test of microbial antagonism between yeasts of the genera Pichia, Hanseníaspora and Meyorozyma versus the fungi Aspergillus niger and Fusarium verticillioides.

[0013] Figure 2 shows an in vitro test of the growth of the fungi Aspergillus niger and Fusarium verticillioides in coffee fruits previously treated with the yeasts Pichia, Hanseníaspora and Meyorozyma.

[0014] Figure 3 shows the layout of the first experiment with fruits under field conditions.

[0015] Figure 4 shows the distribution of fruits by contaminated area according to the treatments and the control referring to the result of the experiment in figure 3 after 20 days.

[0016] Figure 5 shows the graphs relating to the result of the experiment in Figure 3 after 20 days. Petition 870250050109, dated 06 / 16 / 2025, page 8 / 16 / 10 DESCRIPTION OF THE INVENTION

[0017] This invention patent refers to a liquid formulation biological inoculant for coffee fruits that exhibits antifungal action against fungi that are harmful to the fruits.

[0018] The biological inoculant consists of the following formulation: 1L of distilled water; 1x10⁴ - 1x10¹⁰ cells / ml of yeasts of the genera Pichia, Hanseníaspora and Meyerozyma; 1.0% - 10.0% sucrose relative to the volume of solvent; 0.5% - 5.0% yeast extract relative to the volume of solvent; 0.1% - 10.0% propylene glycol relative to the volume of solvent.

[0019] The following yeast species may be used in the formulation of the biological inoculant, the subject of this patent of invention: Meyerozyma amylolytica; Meyerozyma athensensis; Meyerozyma caribbica; Meyerozyma carpophila; Meyerozyma elateridarum; Meyerozyma guilliermondii; Meyerozyma neustonensis; Meyerozyma smithsonii; Hanseniaspora clermontiae; Hanseniaspora gamundiae; Hanseniaspora guilliermondii; Hanseniaspora lachancei Hanseniaspora meyeri; Hanseniaspora occidentalis; Hanseniaspora opuntiae; Hanseniaspora osmophila; Hanseniaspora pseudoguilliermondii; Hanseniaspora uvarum; Hanseniaspora valbyensis; Hanseniaspora vineae; Pichia barkeri; Pichia cactophila; Pichia cecembensis; Pichia cephalocereana; Pichia deserticola; Pichia eremophila; Pichia exigua; Pichia fermentans; Pichia galeiformis; Pichia garciniae; Pichia gijzeniarum; Pichia heedii; Pichia kluyveri; Pichia kudriavzevii; Pichia megalospora; Pichia membranifaciens; Pichia methanothermo; Pichia myanmarensis; Pichia nakasei; Pichia nanzhaoensis; Pichia nongkratonensis; Pichia norvegensis Pichia occidentalis; Pichia paraexigua; Pichia porticicola; Pichia pseudocactophila; Pichia punctispora; Pichia rarassimilans; Pichia scaptomyzae; Pichia scutulata; Pichia sporocuriosa; Pichia terricola; Pichia uvarum.However, it is suggested that the following yeast species be used preferentially: Meyerozyma carpophila, Hanseniaspora uvarum, and Pichia kluyveri. Petition 870250050109, dated 06 / 16 / 2025, page 9 / 16 6 / 10

[0020] In the formulation of the biological inoculant, the subject of this patent of invention, the components act as follows: distilled water acts as a solvent; yeasts act as an antifungal agent; yeast extract acts as a source of nutrients and vitamins for maintaining the yeasts in the formulation; sucrose acts as a carbohydrate source for maintaining the yeasts in the formulation; propylene glycol acts as an emulsifier in the formulation.

[0021] The biological inoculant, the subject of this patent, has proven effective in inhibiting the following genera of fungi in coffee beans: Zygorhynchs, Mucor, Rhízopus, Penicillium, Aspergillus, Trichoderma, Fusarium and Cladosporium. Preparation of the biological inoculant

[0022] The yeasts used in the formulation of the biological inoculant, the subject of this patent, were isolated from the fermentation process of coffee fruits and the colonies were cultivated in YEPG culture medium (15g / l agar, 20g / l glucose, 10g / l peptone and 10g / l yeast extract).

[0023] Yeast cells stored in 2ml microtubes in a -80°C freezer are reactivated in a liquid culture medium containing glucose (20g / l), yeast extract (10g / l) and peptone (10g / l) diluted in 1L of autoclaved distilled water. They are then placed in Erlenmeyer flasks in a ratio of 100 ml of yeast inoculum to 900 ml of culture medium, totaling 1 liter of inoculum. Subsequently, the Erlenmeyer flasks are placed in a shaker at 30°C for 12 hours or until a concentration of 1x10⁹ cells / ml is reached. After this step, the inoculum is poured into cylindrical tubes and centrifuged. This step separates the yeast cells from the culture medium. The supernatant culture medium is discarded and the cells are resuspended in the same volume with a final formulation containing: distilled water; 1.0% - 10.0% sucrose; 0.5% - 5.0% yeast extract; Petition 870250050109, dated 06 / 16 / 2025, page 10 / 16 / 10 0.1% - 10.0% propylene glycol as an emulsifier. In the final step, the composition is packaged in 500 ml bottles, which have been properly sterilized. Finally, the bottles are sealed and stored at 4°C until they are ready for final application. Recommendations for use of the biological inoculant.

[0024] APPLICATION OF BIOCULANT IN THE DRYING PROCESS For small productions, preference is given to the use of manual sprayers of 5l to 20l, for large productions preference is given to the use of motorized sprayers. In both situations, dilute the product in filtered water in a 1:10 ratio and apply to the coffee fruits exposed on a paved or suspended drying patio, in a proportion of 1l of product per 100kg of fruits.

[0025] APPLICATION OF THE BIOLOGICAL INOCULANT IN INDUCED FERMENTATION PROCESS - Preference for applying the product with the composition mentioned in

[0018] , to coffee fruits in a 1:100 ratio, being 1 l of product for 100 kg of fruits, in the open fermentation process; submerged fermentation; carbonic maceration; semi-carbonic maceration or double fermentation. Efficacy tests of the biological inoculant

[0026] Figure 1 demonstrates the ability of the yeasts present in the biological inoculant, the subject of this patent, to antagonize fungi from stored grains that are known to be harmful. The experiment was carried out in Petri dishes containing PDA culture medium, where 10 ml of spore solution of each fungus at a concentration of 1x10⁴ cells / ml were pipetted, specifically at the ends of the plates and a horizontal line in the middle of the plate with 10 ml drops at the concentration of Petition 870250050109, dated 06 / 16 / 2025, page 11 / 16 / 10 1x107 / ml and kept at 28°C for 7 days. Under these conditions, it is noted in Figure 1 that the discs of the cultured fungi were smaller in the plates with the presence of yeasts, and in the cases of treatments with Hanseníaspora and Meyorozyma, a lower density of Aspergillus spores is observed near the line of yeasts.

[0027] Figure 2 demonstrates the performance of a new in vitro assay using petri dishes and agar-water medium, a nutrient-free culture medium, used to position and support coffee fruits that were previously sterilized with a sodium hypochlorite solution and 70% alcohol and subsequently washed with distilled water. This sterilization ensures that the only microorganisms present in the fruit were those selected and artificially added. In the control condition, the sterilized fruits did not receive any inoculation of fungi or yeasts; in the FV condition, 10 ml of spores of the fungus Fusarium verticillioides were inoculated at a concentration of 1x10⁴ spores / ml; in the AN condition, 10 ml of spores of the fungus Aspergillus niger were inoculated at a concentration of 1x10⁴ spores / ml. Under FVMC and ANMC treatment conditions, we sprayed 10 ml of yeast solution of the genus Meyorozyma at a concentration of 1x 107 cells / ml before spraying the fungal spore solutions.Figure 2 shows that the fruits under control conditions remained uncontaminated, demonstrating the efficiency of the sterilization process. We observed that under conditions FV1 and FV3, there was partial fungal growth on the underside of the fruits in contact with the medium, while under condition FV2, there was growth across the entire surface of the fruits. Observing conditions MCFV1 and MCFV3, we concluded that there was a significant reduction in the contaminated area of ​​the fruits compared to FV2, and under condition MCFV3 there was a total reduction in the contaminated area, suggesting that the yeast of the genus *Meyorozyma* has considerable antagonism against the colonizing fungus *Fusarium verticillioides*. Also in Figure 2, we observed that under conditions AN1, AN2, and AN3, the fruits were strongly affected by fungal inoculation; however, the conditions... Petition 870250050109, dated 06 / 16 / 2025, page 12 / 16 / 10 In MCAN1, MCAN2, and MCAN3, we observed a strong effect of the applied yeast, completely inhibiting fungal growth in the fruits and demonstrating that the yeast of the genus Meyorozyma has strong antagonism against the fungus Aspergillus niger, which is one of the main contaminating fungi in the drying and storage process of coffee fruits.

[0028] Based on the results obtained in vitro, we conducted experiments under field conditions to evaluate the performance of the microorganisms. Figure 3 shows the experimental setup using 1 kg portions of ripe coffee fruits separated by bamboo rods in a drying oven with a paved drying floor commonly used for drying coffee fruits, which on sunny days reaches an average temperature above 35°C. These fruits did not undergo sterilization or artificial inoculation with undesirable fungi, so that microorganisms characteristic of the environment could develop. In the treatment conditions, 100 ml of a solution with yeasts of the genera Pichia, Hanseníaspora, and Meyorozyma at a concentration of 1x10⁹ cells / ml were sprayed. In the control condition, 100 ml of boiled water cooled to room temperature was sprayed.

[0029] After twenty days of experimentation, the fruits from each portion were evaluated for contaminated surface area and placed in glass beakers for weighing and visual comparison, as shown in Figure 4. We considered five levels of contamination: 1- Fruit completely free of contamination; 2- Fruit with 1% to 30% contaminated area; 3- Fruit with 31% to 60% contaminated area; 4- Fruit with 61% to 90% contaminated area; 5- Fruit with 90% to 100% contaminated area. We can clearly observe that the number of fruits classified as 1 was higher in all yeast treatments. The number of contaminated fruits classified as 2 and 3, indicating a contaminated fruit area between 1% and 60%, was similar across all treatment and control conditions. However, the Petition 870250050109, dated 06 / 16 / 2025, page 13 / 16 / 10 The number of fruits in classifications 4 and 5, which indicate a contaminated area of ​​the fruits greater than 60%, was significantly lower compared to the control condition. These data are statistically presented in the graphs generated by the Prism software (Figure 5). CONCLUSION

[0030] The biological inoculant, the subject of this patent, has proven to be efficient and effective in inhibiting the growth of deleterious fungi in coffee fruits during the final stages of fruit ripening, storage, drying, and fermentation. Therefore, the biological inoculant can be used as a biological control agent with antifungal action in the pre-harvest and post-harvest phases by coffee producers. Petition 870250050109, dated 06 / 16 / 2025, page 14 / 16

Claims

1 / 2 CLAIM 1. Biological inoculant characterized by the following formulation: 1L of distilled water; 1x10⁴ - 1x10¹⁰ cells / ml of yeasts of the genera Pichia, Hanseníaspora and Meyorozyma; 1.0% - 10.0% sucrose in relation to the volume of solvent; 0.5% - 5.0% yeast extract in relation to the volume of solvent; 0.1% - 10.0% propylene glycol in relation to the volume of solvent.

2. Biological inoculant, according to claim 1, characterized in that the yeasts act as an active ingredient with antifungal action in the formulation.

3. Biological inoculant, according to claim 1, characterized by the fact that the following yeast species are preferably used in the formulation: Meyerozyma carpophyla, Hanseniaspora uvarum and Pichia kluyveri.

4. Biological inoculant, according to claim 1, characterized in that distilled water acts as a solvent in the formulation.

5. Biological inoculant, according to claim 1, characterized in that the yeast extract acts as a source of nutrients and vitamins for maintaining the yeast in the formulation.

6. Biological inoculant, according to claim 1, characterized in that sucrose acts as a carbohydrate source for maintaining the yeasts in the formulation.

7. Biological inoculant, according to claim 1, characterized in that propylene glycol acts as an emulsifier in the formulation. Petition 870250050109, dated 06 / 16 / 2025, page 15 / 16 2 / 2 8. Biological inoculant, according to claim 1, characterized by inhibiting the growth of the following genera of deleterious fungi in coffee fruits: these belonging to the genera: Zygorhynchs, Mucor, Rhizopus, Penicillium, Aspergillus, Trichoderma, Fusarium and Cladosporium.

9. Use of the biological inoculant described in claim 1, characterized by its ability to inhibit the growth of deleterious fungi in coffee fruits during the final stages of fruit ripening, drying, storage and / or induced fermentation. Petition 870250050109, dated 06 / 16 / 2025, page 16 / 16

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