Production, Extraction, and Solubilization of Exopolysaccharides Using Submerged Cultures of Agaricomycetes

Macrofungi, also known as mushrooms, can produce various bioactive compounds, including exopolysaccharides (EPS) with distinct biological properties and subsequent industrial applications in the preparation of cosmetics, pharmaceuticals, and food products. EPS are extracellular polymers with diverse chemical compositions and physical properties secreted by macrofungi in the form of capsules or biofilms into the cellular medium. Submerged cultivation is an industrially implemented biotechnological technique used to produce a wide variety of fungal metabolites, which are of economic and social importance due to their food, pharmaceutical, and agronomic applications. It is a favorable technique for cultivating fungi because it requires little space, minimal labor, and low production costs. Moreover, it allows for control over environmental variables and nutrient supply, essential for the growth of the fungus. Although this technique has been widely applied to yeasts, there is limited knowledge regarding optimal growth conditions for filamentous fungi. Filamentous fungi exhibit different behavior compared to yeast, primarily due to differences in cell morphology, reproductive forms, and the type of aggregates generated during submerged fermentation. Furthermore, various growing conditions can affect the production yield of metabolites, necessitating the development of new knowledge to scale up metabolite production from filamentous fungi. This protocol implements the following culture conditions: an inoculum of three agar discs with mycelium, agitation at 150 rpm, a temperature of 28 °C, an incubation time of 72 h, and a carbon source concentration of 40 g/L. These EPS are precipitated using polar solvents such as water, ethanol, and isopropanol and solubilized using water or alkaline solutions. This protocol details the production procedure of EPS using submerged culture; the conditions and culture medium used are described. A detailed description of the extraction is performed, from neutralization to lyophilization. The concentrations and conditions necessary for solubilization are also described. Key features • Production and extraction of EPS from submerged cultures of mycelial forms of macrofungi. • Modification of the method described by Fariña et al. (2001), extending its application to submerged cultures of mycelial forms of the macrofungi. • Determination of EPS production parameters in submerged cultures of mycelial forms of macrofungi. • EPS solubilization using NaOH (0.1 N). Graphical overview [Image: see text]