Method for producing fungicidal delta-endotoxins by bacillus thuringiensis

Description

10 METHOD FOR PRODUCING FUNGICIDAL DELTA-ENDOTOXINS BY Bacillus thuringiensis Field of invention

[001] The present invention falls within the areas of biotechnology, microbiology and bioprocesses. The invention consists particularly of a culture medium based on forage palm (Nopalea cochenillifera), supplemented with the minerals MnSO4 and K2HPO4 for the production of delta-endotoxins with fungicidal activity against the etiological agents of dermatophytosis caused by Bacillus thuringiensis var. israelensis (Bti). Fundamentals of the invention

[002] Bacillus thuringiensis (Bt) is a Gram-positive, spore-forming bacterium widely used by the biotechnology industry, mainly for bioinsecticide formulations with toxicity to various insect orders, such as Diptera, Lepidoptera, and Coleoptera. Its biological control is mainly due to the production of large crystalline inclusions consisting of entomocidal proteins known as delta-endotoxins. These parasporal crystalline inclusions are composed of several protein structures, called Cry and Cyt, which originate from the stationary growth phase of the bacterium (Xinmin et al., 2023. BMC microbiology, 23, 100).

[003] Despite the use of delta-endotoxins in the control of agricultural pests, their applications are not limited to entomotoxic properties. Recent research indicates that Bt toxins exhibit a variety of biological activities, including nematicidal, antimicrobial and fungicidal properties (Kumar et al., 2021. Egyptian Journal of Biological Pest Control, 31(1), 95; Domínguez et al.,2020. Toxins, 12(7), 430).

[004] Due to their antimicrobial activity, the use of these toxins in the control of pathogenic fungi emerges as a promising alternative for the management of infections caused by dermatophytes in domestic animals. This is because the treatments Petition 870250011858, dated 02 / 13 / 2025, page 8 / 19 / 10 with conventional antifungals, such as terbinafine and azoles like itraconazole and fluconazole, have demonstrated limited efficacy due to fungal resistance and, in addition, prolonged use of antifungals can cause harm to animal health (Lagowski et al., 2020. Infection, 48, 889-897; Begum et al., 2021. Tropical animal health and production, 53, 1-8; Wang et al., 2009. Chinese Journal of Veterinary Medicine 46(11), 1921).

[005] Bt toxins are extremely versatile and widely recognized in the biotechnology sector. It is known that some nutrients present in the culture medium, such as carbon, nitrogen, and micronutrient sources, not only favor the production of delta-endotoxins but also enhance their biological activity (Ozakan et al., 2003. Research in Microbiology, 154, 49-53). Therefore, various fermentative methods with different culture media have been developed for the production of delta-endotoxins.

[006] In this sense, a raw material that can be used as a source of nutrients for the production of delta-endotoxins in Brazil is forage cactus (Nopalea cochenillifera), due to its great abundance in arid and semi-arid regions, occupying an area of ​​approximately 969,589.4 km² (Neves et al., 2021. Biomass Conversion and Biorefinery, 1-12).

[007] In the scientific literature, the biomass of forage palm is described as being composed of approximately 73.8% to 85.2% total carbohydrates and 8.1% to 17.7% mineral matter, with calcium, potassium, and magnesium being the most prominent (Brazil. 2015. EMBRAPA. ISSN 0104866X). The presence of these components provides essential substrates for composing the culture medium for Bacillus, potentially enhancing its efficiency in the production of delta-endotoxins.

[008] Thus, the present invention is characterized as a culture medium based on forage palm supplemented with ammonium sulfate ((NH4)2SO4), manganese sulfate (MnSO4) and dipotassium phosphate (K2HPO4) to produce, in an optimized manner, delta-endotoxins with antifungal activity by Bt var. israelensis (Bti). Petition 870250011858, dated 02 / 13 / 2025, page 9 / 19 / 10

[009] As an example of the use of this substrate, the culture medium based on forage palm extract (Opuntia ficus-indica or Nopalea cochenillifera) as a carbon source, and ammonium sulfate, ammonium chloride or urea as sources of inorganic nitrogen, used to increase the production of biomass, spores and delta-endotoxins with entomotoxic activities, stands out (BR102020002755-7). However, the present invention differs from the aforementioned patent document because, although it uses forage palm as a carbon source and (NH4)2SO4 as a nitrogen source, MnSO4 and K2HPO4 are also added to the culture medium. This formulation of the culture medium results in an increase in the production of delta-endotoxins by Bti, with high fungicidal activity against dermatophytes.

[0010] Similarly, patent document BR 10 2020 017615 3 presents a fermentative method for Bt var. kurstaki using a culture medium composed of cactus biomass (Cereus sp. or Pilosocereus gounellei) as a carbon source and the addition of minerals such as MnSO4 and FeSO4. Although it uses a culture medium where the carbon source is a cactus, the present invention differs from the aforementioned one because it adds (NH4)2SO4 as a nitrogen source and K2HPO4, which increases the production of delta-endotoxins with antifungal activity by Bt. Furthermore, Cereus sp. or Pilosocereus gounellei biomasses are not as bioavailable as forage palm, which facilitates the logistics of the industrial process of the present invention.

[0011] Patents CN1799363 A, GB907030, CN107090420 and CN104046583 demonstrate that agro-industrial by-products or residues such as residual liquor from starch processing, corn starch and beet molasses can be used in Bt cultivation, as well as for the production of delta-endotoxin. Although these materials offer a low-cost substrate, their production is limited to certain regions, since many of these residues are generated locally and on a small scale. In contrast, forage palm stands out as an advantageous alternative, due to its high adaptability to adverse conditions, demonstrating tolerance to prolonged periods of drought, thus allowing its cultivation in various regions, especially in arid and semi-arid climate areas, where other sources would be unfeasible. This makes forage palm substrate a more accessible and economically advantageous resource for the production of culture medium for delta-endotoxins (Werther et al., Petition 870250011858, dated 02 / 13 / 2025, page 10 / 19 / 10 2000. Progress in Energy and Combustion Science, 26(1): 1-27.DOI: 10.1016 / S03601285(99)00005-2; Neves et al., 2021.Biomass ConversionandBiorefinery, 1-12).

[0012] Other inventions concerning Bt production also aim to reduce production costs by using alternative materials. For example, document CN1368549 proposes the use of a fermentation culture medium based on 2% starch, 3% soybean meal, 0.3% baking powder, and 0.1% CaCO3 for the production of toxins with activity for the control of lepidoptera, diptera, and coleoptera. Although also using low-cost substrates, the present patent stands out by adopting forage palm as the main carbon source, along with (NH4)2SO4 as a nitrogen source, in addition to MnSO4 and K2HPO4, forming an efficient cultivation method for the production of toxins specifically targeted at controlling the etiological agents of animal dermatophytosis.

[0013] Another material used for the simultaneous production of delta-endotoxins and proteases is described by patent BR 10 2020 025471 5, the document describes a culture medium based on whey, enriched with minerals such as KH2PO4, MgSO4 and MnSO4, by Bt var. berliner 370 for industrial purposes as a pesticide. However, the patent in question differs by not employing forage palm and (NH4^SO4) in its formulation to produce delta-endotoxins.

[0014] Patent document CN101838624 also describes a fermentation process for obtaining protein crystals produced by Bt AOH-01. The patent proposed a solid microbiological culture medium composed of 1% peptone, 0.5% beef extract, 0.5% sodium chloride, and 1.0-1.5% agar. This process resulted in the production and release of protein crystals used in the control of snails of the genus Oncomelania. However, unlike this invention, which uses a solid medium, the rejected patent advocates a liquid culture medium composed of forage palm, (NH4)2SO4, and the mineral salts K2HPO4 and MnSO4 for the production of delta-endotoxins with fungicidal activity.

[0015] Patent CN104046583 describes a liquid microbiological culture medium based on a vinasse extract solution, prepared with sulfuric acid (0.5-2%) and soybean meal. This culture medium contains, as mineral sources, KH2PO4 (phosphate Petition 870250011858, dated 02 / 13 / 2025, page 11 / 19 / 10 of monobasic potassium), MnSO4 (manganese sulfate), ZnSO4 (zinc sulfate) and CaCO3 (calcium carbonate), which are aimed at producing biomass with high biopesticidal activity. In turn, patent CN107090420 aims at the production of protein crystals and spores of Bt B-Y7-1, through fermentation using soybean meal and corn starch, supplemented with a variety of minerals, including K2HPO4, MnSO4 and ZnSO4, to increase larvicidal activity. Therefore, the present patent differs from the previous ones by employing forage palm as a carbon source, combined with (NH4)2SO4, with the aim of increasing the production of protein crystals by Bti with antifungal activity.

[0016] Another patent application BR1120210251910 describes a fermentative method for the production of spores, protein crystals, and VIP toxin, using the SVBS1801 strain of Bt for the control of the Spodoptera frugiperda pest. The process is carried out in a medium composed of various sources of carbon, nitrogen, and nutrients such as L-glutamine, casein hydrolysate, Bacto casitone, yeast extract, and glycerin, with the addition of salts as a source of minerals: ZnCl2, MgCl2^6H2O, MnClOH2O, CaC^2H2O, FeCl3^6H2O, and 1% HCl. Furthermore, KH2PO4 and K2HPO4 are employed as buffering agents in the fermentative process. Although the culture medium contains yeast extract and L-glutamine, which adequately promote the growth of Bt, their use makes the production process more expensive.However, the present patent differs from the aforementioned one by adopting a simpler composition method, using forage palm, minerals, and ammonium sulfate, which contributes to reducing production costs.

[0017] Other inventions propose the use of inorganic nitrogen sources, such as urea and the amino acid taurine, as reported in patent documents PI 0003314-6 and CN109439603. In contrast, in the present invention, the nitrogen and amino acid source is provided by ammonium sulfate and forage palm, respectively, which results in cost reduction as well as decreased soil leaching and release of ammonia gas into the atmosphere (Camberato, 2017. Purdue University Agronomy Extension, 1-4).

[0018] In view of the foregoing prior art, the present invention is novel, since the omitted elements have not been identified in the literature. Petition 870250011858, dated 13 / 02 / 2025, page 12 / 19 / 10 Description of the invention

[0019] The present invention relates to an innovative fermentation method that uses hydrolyzed forage palm as a carbon source, along with minerals MnSO4, K2HPO4 and (NH4)2SO4 as essential sources of nutrients and nitrogen, respectively, for the cultivation of Bt. This process aims to promote both the increased production of delta-endotoxins by Bt with fungicidal activity and its toxicity against dermatophyte fungi.

[0020] The process omitted in this patent document can be described in more detail below:

[0021] (1) Pre-inoculum: Bt, preferably Bti, should be reactivated and cultivated in a nutrient medium containing organic carbon and nitrogen sources, preferably in Luria-bertani (LB) culture medium composed of 5g / L yeast extract, 10g / L tryptone and 10g / L NaCl, preferably for 18-24 hours.

[0022] (2) Production of the culture medium: The forage cactus (Nopalea cochenillifera), after dehydration and grinding, should be resuspended in a 1% to 3% sulfuric acid solution, preferably 1%, at a ratio of 60 g / L (w / v). The forage cactus suspension in sulfuric acid should be autoclaved at 121°C for 30 minutes and, subsequently, the pH should be neutralized, preferably with a 25% NaOH solution. Finally, the total reducing sugar content should be adjusted to between 5 and 15 mg / mL, preferably 10 mg / mL, and preferably the pH should be adjusted to 7.0. The culture medium should be autoclaved again, preferably for 15 minutes at 121°C.

[0023] According to step (2), sterile minerals at concentrations of 0.015 to 0.045 g / L of MnSO4 and 5 to 15 g / L of K2HPO, preferably at concentrations of 0.0355 g / L and 8.88 g / L, respectively, should be added to the forage palm medium.

[0024] According to step (2), the production medium should be inoculated with 1-50% of the pre-inoculum solution, preferably with an initial cell concentration of Petition 870250011858, dated 02 / 13 / 2025, page 13 / 19 / 10 0.1 g / L (w / v). Then, 0.1% to 0.2% of sterilized (NH4)2SO4 should be added to the hydrolyzed forage palm medium, preferably 0.1%.

[0025] The advantages and production methods of the product can be described more clearly using the following examples: Examples of embodiments of the invention Example 1: Pre-inoculum:

[0026] Initially, Bti was reactivated and cultured in Erlenmeyer flasks with a maximum capacity of 250 mL containing 100 mL of LB (Luria-Bertani) culture medium, where it was kept for 18 hours in a rotary incubator at 200 rpm and 30°C. Example 2: Evaluation of delta-endotoxin production:

[0027] Initially, the forage cactus (Nopalea cochenillifera) was subjected to a sanitization process using sodium hypochlorite. Then, the cactus was cut into small pieces and placed in a drying oven at a temperature of 60 °C. After complete dehydration, the pieces were ground in an industrial blender, resulting in a powder. This powder was then mixed with a 1% sulfuric acid solution at a concentration of 6% (w / v). The resulting suspension was autoclaved at 121°C for 30 minutes and its pH adjusted to 7.0. Subsequently, the culture medium was centrifuged at 3500 rpm for 10 minutes, and the total reducing sugar content was adjusted to 10 mg / mL and autoclaved for 15 minutes at 121°C. After autoclaving, the pre-inoculum (as described in example 1) was added to the culture medium, obtaining an initial cell concentration of 0.1 g / L (w / v).In addition, 0.1% of (NH4^SO4), previously sterilized by filtration, was incorporated, along with 0.0355 g / L of MnSO4 and 8.88 g / L of K2HPO4.

[0028] Delta-endotoxins produced after 72h of fermentation were solubilized in 50mM NaOH solution and determined by protein quantification using the BCA™ Protein Assay Kit (Thermo Scientific) (Yudina et al., 2007. Anaerobe, 13(1), 6-13; Cahan et al., 2008. Microbiology, v. 154 (11), 3529-3536). The addition of salts resulted in a final production of delta-endotoxins superior to that of the LB culture medium. Petition 870250011858, dated 02 / 13 / 2025, page 14 / 19 / 10 (Luria-Bertani) and to the culture medium based on forage palm in the absence of MnSO4 and K2HPO4, indicating a higher yield than the forage palm medium without supplementation and similar to an industrial culture medium, as shown in the table below:

[0029] Table 1: Delta-endotoxin production in different culture media Delta-endotoxin culture medium (g / mL) MPF 168.44 ± 0.15 MPF + MnSO4e K2PO4 504.00 ± 0.15 LB 690.11 ± 0.09

[0030] MPF: Culture medium based on forage palm without mineral salt supplementation. MPF + MnSO4 and K2PO4: Culture medium based on forage palm supplemented with (NH4^SO4 and MnSO4 and K2PO4). LB: Luria-Bertani culture medium. Example 3: Evaluation of the antifungal activity of delta-endotoxins:

[0031] Bti biomass cultivated in forage palm medium with MnSO4 and K2HPO4 was washed with 1 M sodium chloride solution and ice-cold distilled water, centrifuging after each wash. The precipitate was resuspended in 50 mM sodium hydroxide solution and incubated at 37°C for 1 h to solubilize the delta-endotoxins. After incubation, the suspension was centrifuged at 5000 rpm for 10 minutes, and the concentration of delta-endotoxins was determined using the BCA™ Protein Assay Kit (Thermo SCIENTIFIC). The pH was adjusted to 8.8 using Tris-HCl buffer with pH 6.5. Then, trypsin was incorporated into the solution in a 1:100 ratio (enzyme:delta-endotoxin; p:p). The mixture was kept at 37°C for 1 Petition 870250011858, dated 02 / 13 / 2025, page 15 / 19 / 10 hour, and the reaction was completed with the addition of PMSF at a final concentration of 10 mM and urea at 2 mM (Yudina et al., 2007. Anaerobe, 13(1), 6-13; Cahan et al., 2008. Microbiology, v. 154 (11), 3529-3536).

[0032] After extraction and activation of the toxins, the samples were lyophilized and resuspended in RPMI 1640 culture medium, and concentrations of 0.002 μg / ml to 1.04 μg / ml were prepared. Subsequently, the material was filtered through a membrane with a porosity of 0.22 µm (Millipore) and used to evaluate susceptibility activity against the dermatophytes Microsporum canis, Microsporum gypseum, and Trichophyton mentagrophytes var. mentagrophytes. The procedure adopted for the evaluation of fungal susceptibility was developed according to the broth microdilution technique, using the standardization of protocol M-38 A2 (2008) by the Clinical and Laboratory Standards Institute (CLSI).

[0033] Dermatophyte samples were seeded in test tubes containing oat agar and incubated in a microbiological growth incubator at 30°C until conidia were obtained. After sporulation, 5 mL of sterile 0.85% saline solution were added to the tubes and, using a sterile disposable loop, the spores and hyphae were gently detached. The suspension was adjusted to 70% transmittance with an optical density of 530 nm. Finally, the samples were diluted in a 1:50 ratio in RPMI-1640 medium to obtain a suspension twice as concentrated as the density required for the test, corresponding approximately to (1x10³ to 3x10³ CFU / mL).

[0034] The procedure was performed in duplicate and the samples were distributed in microtiter plates, according to the standardization of the aforementioned protocol to determine the minimum inhibitory concentration and the minimum fungicidal concentration of the endotoxins (tables 2 and 3).

[0035] Table 2. Minimum inhibitory concentration of delta-endotoxins from Bacillus thuringiensis var. israelensis produced in hydrolyzed forage palm medium with the addition of (NH4)2SO4 and the minerals MnSO4 and K2HPO4, against newly isolated animal dermatophytosis. Petition 870250011858, dated 13 / 02 / 2025, page 16 / 19 / 10 Dermatophyte fungi MIC (pg / mL·1) MFC100 (pg / mL-1) Trichophyton mentagrophytes ATCC 6272 Trichophyton mentagrophytes 0.13 0.13 0.26 0.26 Microsporum canis ATCC 6966 0.06 0.06 Microsporum canis 0.016 0.016 Microsporum gypseum ATCC 6921 0.13 0.26 Microsporum gypseum 0.06 0.23

[0036] MIC (pg / mL-1): Minimum inhibitory concentration. MFC100 (pg / mL-1): Minimum fungicidal concentration.

[0037] Table 3. Minimum inhibitory concentration of delta-endotoxins from Bacillus thuringiensis var. israelensis produced in Luria Bertani culture medium. Dermatophyte fungi MIC (pg / mL-1) CFM1oo(pg / mL-1) Trichophyton mentagrophytes ATCC 6272 1.96 3.92 Trichophyton mentagrophytes 1.96 1.96 Microsporum canis ATCC 6966 0.98 1.96 Microsporum canis 0.98 1.96 Microsporum gypseum ATCC 6921 0.98 1.96 Microsporum gypseum 0.98 1.96

[0038] MIC (pg / mL-1): Minimum inhibitory concentration. CFMioo (pg / mL-1): Minimum fungicidal concentration. Petition 870250011858, dated 02 / 13 / 2025, page 17 / 19

Claims

CLAIMS 1. Method for producing Delta-endotoxin Fungicidal by Bacillus thuringiensis, characterized by being composed of hydrolyzed forage palm, supplemented with ammonium sulfate and minerals, in specific proportions, intended to increase the fungicidal activity of the delta-endotoxins produced.

2. Method for producing Delta-endotoxin Fungicidal by Bacillus thuringiensis, according to claim 1, characterized by using ammonium sulfate as a nitrogen source and the minerals MnSO4 and K2HPO4 as sources of essential nutrients.

3. Method for producing Delta-endotoxin Fungicidal by Bacillus thuringiensis, according to claim 1, characterized in that the sulfuric acid from the hydrolysis of forage palm is in a concentration range of 1% to 3% and the total reducing sugars are in a range of 5 to 15 mg / ml.

4. Method for producing Delta-endotoxin Fungicidal by Bacillus thuringiensis, according to claim 1, characterized by the concentration of MnSO4 and K2HPO4 varying in a range between 0.015 to 0.045 g / L and 5 to 15 g / L respectively.

5. Method for producing Delta-endotoxin Fungicidal by Bacillus thuringiensis, according to claim 1, characterized by being a liquid fermentative process composed of a carbon source, nutrients and nitrogen. Petition 870250011858, dated 13 / 02 / 2025, pp. 18 / 19