Filippe Elias de Freitas Soares

Proteases are widely studied hydrolases as “green technologies” for degrading structures within complex matrices, although combining different classes poses challenges for catalytic stability. To the best of our knowledge, this is the first study to specifically evaluate the biochemical control of Cryptosporidium spp. oocysts using plant- and microbial-derived proteases. This study evaluated the compatibility and sanitizing potential of the microbial serine protease subtilisin Carlsberg (HPF-1SCA) and the plant cysteine protease papain (1PPP), both individually and in a 15% (w/v) combination, hypothesizing a synergistic effect due to their distinct catalytic specificities, as a potential biochemical approach to reduce contamination by these zoonotic protozoa of global importance, which are highly resilient to conventional disinfectants. The experimental design comprised five distinct treatment groups: a negative control (distilled water), a positive chemical control (0.04% v/v NaClO), treatment with isolated papain (15% w/v), treatment with isolated microbial HPF formulation (15% w/v), and a combined treatment using both enzymes simultaneously (15% w/v each). Concurrently, an in silico molecular docking investigation was conducted to elucidate the predicted binding scores, structural compatibility, and preferential binding mechanisms of these enzymes toward the Cryptosporidium Oocyst Wall Protein (COWP). In vitro compatibility assays revealed an immediate antagonistic effect due to mutual proteolysis, reducing overall proteolytic activity by 45% after 72 h. Despite this antagonism, the enzyme mixture (G5) and the isolated microbial protease (G4) achieved a 93% reduction in oocysts, equivalent to the conventional 0.04% NaClO treatment (G2), while papain (G3) achieved 65%. All these results represented statistically significant efficacy (p < 0.01) compared to the negative control (G1). In silico molecular docking studies provided a structural predictive basis for the experimental data, suggesting that subtilisin Carlsberg (HPF-1SCA) exhibits more favorable predicted binding scores and structurally compatible interfaces, compared to papain, particularly toward the major oocyst structural proteins COWP8 (estimated ΔG = −15.2 kcal·mol⁻¹) and COWP6 (estimated ΔG = −13.3 kcal·mol⁻¹). This provides robust evidence for initial target recognition and molecular anchoring, although these static models do not definitively confirm catalytically productive cleavage conformations. In summary, this in vitro proof-of-concept demonstrates that microbial proteases show promise for the biochemical destabilization of oocysts without generating toxic chlorinated byproducts. While these baseline findings align with the One Health concept, future field-scale validations assessing enzyme stability under variable environmental conditions and in vivo infectivity assays are required before practical application in sanitation protocols.

Nematophagous fungi are important biological control agents that naturally reduce nematode populations through specialized predatory mechanisms. This study evaluated the viability and predatory capacity of the nematophagous fungus Duddingtonia flagrans (AC001) after fourteen years of refrigerated storage in sodium alginate pellets. Fungal viability was assessed by reactivating the pellets on Corn Meal Agar and Potato Dextrose Agar, with subsequent monitoring of mycelial growth and morphological confirmation by light microscopy. The predatory capacity was determined by means of in vitro assays using Panagrellus redivivus as a model organism. Plates previously colonized by the fungus received approximately 400 nematodes and were compared with fungus-free controls. Predatory activity was evaluated daily for five days by counting live nematodes in random microscopic fields. Final recovery of viable nematodes was performed using a modified Baermann technique. The data were analyzed using Student’s t-test (p < 0.05). Isolate AC001 exhibited successful reactivation, preserved morphological characteristics, and sustained high predatory activity, resulting in reductions in nematode populations ranging from 91% on the first day to 95% on subsequent days, with a final reduction of 67% in nematode recovery. These findings demonstrate that sodium alginate encapsulation combined with refrigeration enables the exceptionally long-term preservation of functional and predatory attributes of nematophagous fungi, supporting its applicability in biological control strategies.

Agriculture is fundamental to food security, but it faces challenges such as plant-parasitic nematodes, which cause significant economic damage. Chemical control, although effective, can lead to pathogen resistance, environmental impacts, and health risks. This study evaluated the proteolytic activity of three commercial protease formulations from Bacillus licheniformis (APP 1500, HPP, and HPF) in the control of Meloidogyne incognita in vitro and under greenhouse conditions. Activity was measured at pH 8.0 and 50°C for 15 minutes. In the in vitro test, the following experimental groups were set up: negative control (water) and treatments containing the products at concentrations of 1% and 5% (w/v). In the greenhouse experiment, the treatments included the commercial protease formulations, a negative control (water), and a positive control (Vertimec®). The in vitro test showed a significant reduction in the number of eggs, especially at the 5% concentration: 42% (APP 1500), 70% (HPP), and 56% (HPF). In the greenhouse, all the enzymes significantly reduced the number of eggs and galls, with efficiency exceeding 80% for APP 1500 and HPP, and exceeding 90% for HPF, with no statistically significant difference compared to the chemical control. The tests also showed that the proteases’ catalytic activity is maintained even under high pH and temperature conditions. These results indicate that the enzyme products have promising potential as a sustainable alternative to chemical nematode control.

Monieziosis presents a considerable challenge to livestock farming, mainly due to the parasite’s resistance to common anthelmintics, prompting the need for alternative control strategies. This study examined the in vitro effect of two enzymatic sources (plant and fungal) on the structural integrity of Moniezia expansa eggs, aiming to find new environmental management solutions. The plant enzyme papain was tested at various concentrations and time points, while the fungal enzyme was produced through solid-state fermentation using Duddingtonia flagrans (AC001), resulting in an active crude enzymatic extract (ACEE). Papain at 10% w/v showed nonlinear degradation (R² = 0.998), achieving 95% egg reduction after 48 h. The fungal extract ACEE (71.2 U mL⁻¹) caused a 60% reduction after 72 h. Morphological studies indicated significant eggshell damage following both treatments. A compatibility assay showed an antagonistic interaction, with enzyme activity decreasing by 83.83% within 48 h, likely due to cross-proteolysis. Although each agent is effective individually, combining them is not feasible. This is the first study documenting the activity of ACEE and papain against M. expansa eggs, and it recommends using them separately or sequentially for effective parasite control.

Ascaris lumbricoides is one of the most epidemiologically significant soil-transmitted helminths, and the environmental persistence of its eggs is largely attributed to their robust structural architecture. The search for ovicidal alternatives capable of overcoming this barrier has increasingly focused on metallic nanoparticles obtained through biological synthesis. Scanning electron microscopy (SEM) was employed to evaluate the ultrastructural effects of silver nanoparticles (AgNPs) biosynthesised by the nematophagous fungus Duddingtonia flagrans on A. lumbricoides eggs. Ultraviolet-visible spectroscopy and transmission electron microscopy confirmed the synthesis of AgNPs, revealing predominantly spherical, well-dispersed particles with an average diameter of 9.22 ± 4.9 nm. Cytotoxicity assays indicated an IC₅₀ of 7.7 µg/mL. SEM analyses showed that eggs in the control group maintained intact morphology, with no apparent deformities. In contrast, exposure to AgNPs induced pronounced structural alterations, including marked wrinkling, surface erosion and shell collapse, suggesting disruption of multiple layers. Albendazole alone produced deep linear fissures consistent with internal metabolic failure, though with minimal external erosion. The combined treatment with AgNPs and albendazole resulted in severe degradation. These findings demonstrate that AgNPs exhibit significant ovicidal activity and may serve as effective adjuvants to enhance the action of conventional anthelmintics against highly resistant helminth eggs.

The use of proteolytic enzymes in association with entomopathogenic fungi offers a promising alternative for improving the biological control of insect pests. This study evaluated the compatibility between Beauveria bassiana and papain and the effectiveness of their combined application in controlling Tenebrio molitor. Conidial viability in the presence of papain was monitored for 48 h and showed a reduction in germination from 100% to approximately 70%, without detrimental effects on fungal performance. Papain activity remained stable up to 12 h, declining afterward, indicating biochemical compatibility. Bioassays revealed significant differences among treatments (p < 0.01). In larvae, mortality ranged from 5.18 ± 0.19% in the control to 49.62 ± 2.00% with papain, 62.24 ± 0.58% with conidia, and 89.71 ± 1.06% in the combined treatment; papain and conidia alone did not differ statistically. In pupae, mortality reached 2.20 ± 0.00% in the control, 47.38 ± 0.69% with papain, 63.69 ± 0.69% with conidia, and 85.91 ± 0.84% with the combination, with all treatments differing significantly. Fungal reisolation confirmed typical B. bassiana development. Overall, the results show that papain does not compromise fungal viability and that its combination with B. bassiana enhances entomopathogenic activity, supporting its potential for integrated pest management.