Background

Macrofungi are fungi that produce reproductive bodies that are visible to the naked eye. Some macrofungi known for their production of polysaccharides are the Shiitake (Lentinula eodes) or the mushroom turkey tail (Trametes versicolor). Macrofungal polysaccharides are a group of compounds of great interest in bioprospecting studies of both medicinal and edible fungi, owing to their broad spectrum of bioactivities, including cytotoxic, antiproliferative, antitumor, immunomodulatory, anti-inflammatory, antioxidant, and stimulatory effects, on the colon microflora (Arora and Tandon, 2015; X. Meng, 2016; Nowakowski et al., 2021). These bioactivities are related to the chemo-preventive action of macrofungal polysaccharides against different types of cancers such as colorectal cancer and other chronic diseases. Macrofungi polysaccharides are complex carbohydrates found in the cell walls and extracellular matrices. These polysaccharides play a crucial role in the structural integrity and functionality of fungal organisms. They can be homopolysaccharides that contain repeating units of the same monosaccharide or heteropolysaccharides, containing several types of monosaccharides. Polysaccharides can also include other groups, such as sulfate or glucuronate, and adopt simple, triple, or random helical conformations depending on the conditions of the culture medium (Huang and Nie, 2015; Gong et al., 2020), significantly affecting their solubility in water or marginally basic aqueous solutions. Solubility and the type of conformation adopted are crucial for biological activity and must be controlled in different assays at the in vitro level because of their effects on the expected results. Structural conformation is also responsible for the great water solubility of polysaccharides: for example, scleroglucan secreted by Sclerotium fungi adopts a highly ordered, rigid, triple helical tertiary structure when dissolved in water at room temperature and low concentrations of alkali, usually below 0.15 M NaOH (Castillo et al., 2015). Water solubility of fungal exopolysaccharides (EPS) depends, to a large extent, on monosaccharide composition and structure and size of the respective molecules (Jaros et al., 2018). These variables, together with differences in the type of bonding and branching, tertiary structure, electrical charge and conformation, and solubility generate a diversity of physical and chemical properties, as well as reported biological activities (Bacic et al., 2009).

Polysaccharides produced by macrofungi can be classified into three main groups based on their location in the cell: (1) cytosolic polysaccharides that provide a source of carbon and energy for the cell; (2) cell wall polysaccharides that include the peptidoglycans, teichoic acids, and lipopolysaccharides; and (3) extracellular polysaccharides that are exuded in the form of capsules or biofilms, also known as EPS, which constantly diffuse into the cell wall and cell cultures (Kambourova et al., 2015; Wang et al., 2022). For the production of EPS, the submerged culture technique is used at both laboratory and industrial scales, where the mycelium of the fungus grows while floating as small spheres called pellets when there is agitation (Papagianni, 2004). The submerged fermentation process is widely used for producing different types of metabolites on a large scale or under flask conditions in which organisms can be obtained in limited physical spaces under conditions controlled and optimized for the production of biomass; another advantage is the ease of separating the mycelium from the EPS in the culture medium, obtaining a product of uniform quality for their purification in a further step. On the other hand, it provides several advantages such as higher productivity and yields, lower labor costs, and lower contamination risk. Submerged culture offers the potential advantages of faster production of EPS within a smaller space, reduced possibility of contamination, and better control of cultivation conditions such as carbon source and temperature (Dudekula et al., 2020).

In addition to submerged cultures, several other methods have been employed for the production of EPS. Some of these methods are: (1) surface culture, in which the microorganisms are grown on the surface of a solid substrate, such as agar or other suitable media; the EPS is produced and secreted by the microorganisms onto the surface of the substrate (Breitenbach et al., 2022); (2) solid-state fermentation, which involves the growth of microorganisms on solid substrates without the addition of free-flowing water; the microorganisms utilize the nutrients present in the solid substrate to produce EPS; common solid substrates used include agricultural residues, cereal grains, and sawdust (Isikhuemhen et al., 2014); (3) biofilm reactors, which provide an ideal environment for the growth of biofilms, allowing the microorganisms to attach to a surface and form a structured matrix; EPS production occurs with the biofilm structure (Seneviratne et al., 2008); (4) immobilized cell systems, in which the microbial cells are immobilized or attached to a solid support matrix, such as alginate beads, polyurethane foam, or glass beads; the immobilized cells produce EPS while attached to the support matrix, allowing for easy separation and recovery of the EPS (Li et al., 2017); and (5) membrane bioreactors, which combine the use of submerged culture with the retention of microbial cells and EPS using a membrane filtration system; the membrane allows the culture medium to pass through while retaining the microbial cells and EPS, which can then be harvested and recovered (Brito et al., 2019). It is important to note that the method of choice for EPS production depends on various factors, including the microorganism used, the type of EPS desired, and the scale of production. Different methods offer distinct advantages and disadvantages in terms of yield, productivity, cost, and ease of operation.

Extensive investigations have been conducted to enhance the yield of EPS production. These studies have focused on optimizing various factors, including the assessment of different carbon sources (e.g., glucose, sucrose, fructose) and nitrogen sources (e.g., peptone, yeast extract, ammonium nitrate). The influence of pH levels and incubation time on scleroglucan production has also been thoroughly examined. Notably, Fariña et al. (2001) successfully identified the optimal conditions for scleroglucan production by Sclerotium rolfsii. Their research determined the most advantageous carbon and nitrogen sources, optimal pH range, and incubation time, resulting in significant improvements in scleroglucan yields. Our approach differs from the method described by Fariña et al. (2001), as we evaluate the impact of two carbon sources. Moreover, we consider the contribution of the mushroom species as a crucial factor in EPS production. Incorporating a comprehensive range of tests during initial evaluation enables the identification of optimal strains for bioactive EPS production (Mahapatra and Banerjee, 2013; Angelova et al., 2022). Among agaricomycetes, Agaricales and Polyporales are the two most prominent and extensively used orders in biotechnology. To validate this protocol, representative strains from Agaricales and Polyporales were selected, namely Schizophyllum radiatum and Lentinus crinitus, respectively.