Co‐cultivation and Medium Optimization: A Strategy for Discovering Fungal‐derived Protease Inhibitors

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Abstract
The fungi Fusarium guttiforme and Phytophthora palmivora were cultivated in four different media (Potato Dextrose Agar, Czapek, rice, and ISP2) and co‐cultured to stimulate fungal interactions and enhance secondary metabolite production. Promising extracts were fractionated, yielding compounds such as the iron complex of fusaric acid ( 1 ), magnesium complex of fusaric acid ( 2 ), haematocin ( 4 ), fusarinolic acid ( 7 ), and cyclonerodiol ( 8 ). These compounds exhibited significant papain inhibitory activity, with compounds 2 and 4 showing IC 50 values below 20 µM. Notable anti‐ Trypanosoma cruzi activity was also observed, particularly for compounds 2 (50% inhibitory concentration [IC 50 ] = 12.19 µM) and 4 (IC 50 = 13.32 µM). Additionally, antifungal activity against Candida spp. was detected, with compound 2 and the magnesium complex of 9,10‐dehydrofusaric acid complex ( 3 ) showing MICs of 50 µg/mL. This study highlights the potential of fungal co‐cultivation and variation in culture medium as effective strategies for discovering novel bioactive protease inhibitors and antiparasitic agents.

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