Utilizing agricultural waste to produce mushrooms may be a cost-effective and environmentally friendly proposition to address the nutritional and health demands of the growing global population. Mushrooms can grow on a range of substrates and their selection is based on their availability and cost. In this study, five types of local waste were mixed: olive crop residues (OC), coffee residue (CR) or rice husk (RH) with wheat straw (WS) and beech wood shavings (BW), respectively. Then, the mixtures were sprayed with 20% w/w lipid fermentation wastewater (LFW) from Rodosporidium toruloides that was used as an alternative substrate-moistening method. Afterwards, these mixtures were tested for cultivating Pleurotus spp., Ganoderma spp. and Lentinula edodes. The results showed that the substrate significantly affected the incubation period and the biological efficiency (BE), with OC mixed substrates proving to be the most favorable across the different species. Pleurotus spp. had the shortest cultivation times and the highest BE, while G. lucidum required the longest incubation periods and had the lowest BE, particularly on CR substrates. The study also found that substrates affected mushroom morphology. Nutritional analysis revealed significant differences in protein, polysaccharides, lipids, ash and energy content, depending on the species and substrate. High protein levels were found in P. eryngii (28.05–29.58% d.w.) and G. resinaceum (28.71–29.90% d.w.). The elevated total phenolic compounds (28.47–40.17 mgGAE/g) values in carposomes from CR and OC substrates for Ganoderma spp., L. edodes, P. pulmonarius and P. ostreatus, along with antioxidant activity (DPPH, ABTS, FRAP) assays, highlighted the crucial role of substrate composition in enhancing the medicinal properties of mushrooms. The mixed substrates also influenced the fatty acid (FA) and polysaccharide composition, with WS increasing unsaturated FAs and glucose (<69.8%) being the primary monosaccharide. The study suggests that using the spraying method of 20% w/w LFW as a moisture agent in these substrates is effective for mushroom production.
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