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This study evaluated the production yield of fungal chitosan (FuCho) by nine Mucorales strains, including novel species isolated from Brazilian biomes, cultivated in alternative culture media composed of corn steep liquor (CSL) and biodiesel-derived glycerol (GLY). Furthermore, the physicochemical properties, potential of irritation, and broad-spectrum antimicrobial activity of the extracted FuCho were investigated. Among the strains tested in CSL-GLY medium, FuCho yields ranged from 17.88\u00a0mg\/g (Absidia aguabelensis) to 126.56\u00a0mg\/g (Absidia caatinguensis). Optimization using a 22 central composite rotatable design (CCRD) yielded maximum FuCho productions of 128.26\u00a0mg\/g for A. caatinguensis (center point condition) and 164.92\u00a0mg\/g for Cunninghamella elegans (lower CSL concentration). Physicochemical characterization revealed degrees of deacetylation of 85% (A. caatinguensis) and 75% (C. elegans), crystallinity indices of 36.74% and 37.93%, and viscometric molecular weights (Mv) of 1.6\u00d7103\u00a0g\/mol and 2.0\u00d7103\u00a0g\/mol, respectively. In the HET-CAM assay, both FuCho samples were classified as non-irritating, exhibiting no inflammatory, vascular, or vasoconstrictive effects. FuCho demonstrated minimum inhibitory concentrations (MIC) against all tested pathogenic bacterial strains and Candida species, with A. caatinguensis FuCho displaying lower MIC values for the majority of microorganisms compared to C. elegans FuCho. Confocal laser scanning microscopy confirmed a significant reduction in microbial cell viability, driven by membrane disruption in Escherichia coli and Staphylococcus aureus. These findings validate the CSL-GLY mixture as a cost-effective, eco-friendly culture medium for producing high-value biopolymers with pharmaceutical potential, underscoring the biotechnological relevance of Brazilian Mucorales strains.<\/jats:p>","DOI":"10.3390\/molecules31162807","type":"journal-article","created":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T11:02:11Z","timestamp":1786532531000},"page":"2807","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Valorization of Corn Steep Liquor and Glycerol for Fungal Chitosan Production by Mucorales from Brazilian Biomes: Structural Characterization and Antimicrobial Activity"],"prefix":"10.3390","volume":"31","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8801-3361","authenticated-orcid":false,"given":"L\u00facia Raquel Ramos","family":"Berger","sequence":"first","affiliation":[{"name":"Departamento de Laborat\u00f3rios Multiusu\u00e1rios, Campus Universit\u00e1rio Sede, Universidade Federal do Agreste de Pernambuco, Av. 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