The carbon-to-nitrogen (C/N) ratio in the cultivation medium significantly influences the growth rate, vigor of mycelium, yield of fruiting bodies, and their nutritional composition. Recently, agricultural and forestry wastes have been increasingly used in cultivating Flammulina velutipes. However, systematic research on how these materials affect the nutritional and functional properties of the fruiting bodies is lacking. This study investigated the effects of different C/N ratios on F. velutipes cultivation. We evaluated the agronomic traits, nutritional composition, and flavor compounds of the fruiting bodies. Our findings reveal that an optimal C/N ratio of 27:1 in the composted substrates enhances the total yield of fruiting bodies, with 25.1% soybean straw as the primary raw material. This ratio also significantly increases the levels of crude protein, total amino acids, and essential amino acids in the fruiting bodies (p < 0.05). Fruiting bodies from the high-nitrogen (HN) treatment showed the highest content of umami amino acids and equivalent umami concentration value. Additionally, we employed an untargeted liquid chromatography–mass spectrometry (LC-MS)-based metabolomics approach to analyze the metabolite profiles of fruiting bodies cultivated in high-nitrogen (HN), medium-nitrogen (MN), and low-nitrogen (LN) substrates. We found that the carbon–nitrogen ratio can affect the flavor and quality of fruiting bodies by regulating amino acid biosynthesis and metabolism and other related pathways. Our results suggest that a C/N ratio of 27:1 offers numerous benefits for the cultivation of F. velutipes with comprehensive analyses and has promising application prospects.

Recently, there has been a growing interest in using agricultural and forestry residues to cultivate Flammulina filiformis. However, there is limited research on cultivating F. filiformis with soybean straw as a substrate. This study systematically optimized the cultivation formula for F. filiformis using soybean straw as the raw substrate and explored the effects of the water content, carbon-to-nitrogen ratio (C/N ratio), substrate particle size, and substrate loading on its growth and development. By replacing corncob, wheat bran, and soybean hulls with soybean straw and increasing the proportion of rice bran, the cultivation formula for growing F. filiformis was optimized. We found that the maximum fruiting body yield of 405 g (330 g dry substrate per bottle) and a biological efficiency of 122.73% were achieved using a substrate mixture of 25% soybean straw, 20% corncob, 20% cottonseed hull, 25% rice bran, 8% wheat bran, 1% CaCO₃, and 1% shellfish powder. The yield and biological efficiency of fruiting bodies cultivated on the substrate containing 25% soybean straw did not show significant differences compared to the control group. However, the cultivation formula containing 25% soybean straw yielded F. filiformis with significantly higher levels of amino acids, essential amino acids, and fat. These findings suggest that the 25% soybean straw substrate formulation can serve as a viable alternative to the control formulation for the cultivation of F. filiformis, although variations in the nutritional composition exist. Based on this optimized formula, an optimal biological efficiency can be achieved with a substrate-to-water ratio of 1:1.7, a wet substrate loading amount of 940 g (in a 1250 mL cultivation bottle), and a soybean straw particle size range of 6–8 mm. The optimal C/N ratio for cultivating F. filiformis using soybean straw ranges from 27:1 to 32:1. Additionally, orthogonal experiments revealed that the nitrogen content significantly affected the fruiting body yield, stipe length, and stipe diameter, while the water content mainly affected the pileus diameter, pileus thickness, and number of fruit bodies. Under defined conditions (dry substrate loading volume of 337 g (in a 1250 mL cultivation bottle), a substrate-to-water ratio of 1:1.6, and a C/N ratio of 26:1), the maximum yield and biological efficiency per bottle reached 395 g and 117.21%, respectively. Our findings indicate that the F. filiformis cultivation using soybean straw as the raw substrate exhibits a promising performance and extensive application potential.