Bioscience Evidence 2026, Vol.16, No.4, 316-328 http://bioscipublisher.com/index.php/be 320 only related to nutrient composition but also associated with fiber structure, aeration capacity, and water-holding ability of the materials. 3 Substrate Management During Shiitake Mushroom Bag Production 3.1 Substrate preparation, bag filling, and sterilization management Shiitake mushroom bag production usually uses lignocellulosic materials such as sawdust or agricultural and forestry residues as the basic substrate components. Nutrient supplements, including wheat bran, grain powder, and mineral elements, are added according to the nutritional requirements of mycelial growth. The substrate preparation process generally includes raw material crushing, mixing, moisture adjustment, supplementation with additives, bag filling, and sterilization. The main purpose of these steps is to create suitable nutritional and physical conditions for shiitake mycelial growth. Insufficient moisture can restrict mycelial expansion, while excessive moisture may reduce aeration and increase the risk of contamination. In most bag cultivation systems, raw materials need to be fully moistened and adjusted before bag filling to maintain an appropriate moisture level. Shanmugaraj et al. (2024) evaluated different agricultural by-product substrates for shiitake cultivation in West Bengal, India. The tested materials included sawdust, rice straw, wheat straw, corn cob, sugarcane bagasse, and bean shells. After soaking, the moisture content was adjusted to 60%-65%, and wheat bran and CaCO₃ were added for nutritional and structural regulation, forming a typical substrate preparation process based on agricultural residues. Kumar et al. (2022) also applied a combination of moisture adjustment, mineral supplementation, and sterilization in agricultural waste-based substrates, indicating that whether traditional sawdust systems or alternative agricultural residue systems are used, maintaining substrate stability and reducing contamination risks are essential foundations for successful bag cultivation (Figure 2). In commercial production, high-temperature sterilization is commonly used to treat substrates by reducing competition from bacteria, molds, and other microorganisms, thereby improving bag cultivation success rates. Because different substrate materials vary in structure and moisture status, sterilization efficiency depends not only on temperature and treatment duration but also on substrate density, particle size, and bag compaction level. Figure 2 Experimental steps for Shiitake mushroom cultivation using agro-industrial wastes (Adopted from Kumar et al., 2022) 3.2 Mycelial colonization and bag maturation management After inoculation, shiitake mushroom bags first enter the vegetative growth stage. The mycelia begin to utilize lignocellulosic materials and nutrient supplements in the substrate and gradually complete the maturation process, including substrate colonization, mycelial film formation, mycelial knot development, browning, and formation of the outer protective mycelial layer. For growers, a fully colonized bag does not necessarily indicate that
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