Spent mushroom substrate (SMS) is the main co-product generated during the cultivation of Pleurotus ostreatus. This by-product is currently used almost exclusively for composting, and thus represents a significant management and environmental challenge for mushroom growers. However, SMS is rich in dietary fiber, making it a promising matrix for the recovery of bioactive compounds, particularly polysaccharides with potential functional properties. In this context, conventional extraction methods are being progressively replaced by more efficient and sustainable emerging technologies, such as ultrasound-assisted extraction (UAE), which relies on acoustic cavitation to disrupt cellular structures, enhance mass transfer, and increase extraction yields. Therefore, the present study aims to optimize the extraction of soluble dietary fiber (SDF) from SMS of Pleurotus ostreatus using UAE.

The response surface method (RSM) combined with the Box–Behnken design method (BBD) was used to optimize the content of SDF. The effects of the solid–liquid ratio (15–40 g/L), ultrasound power (200–500 J/mL), and particle size (0.25–2 mm) on the content of SDF were examined. The experimental data obtained were fitted to a second-order polynomial equation using multiple regression analysis and analyzed by Statgraphics 19.0. It was found that two factors represented a significant effect (ratio and particle size). The coefficient of determination (R²) for the model was 80.8%. The optimal conditions for the extraction of SDF were a 27 g/L solid–liquid ratio, 200J/mL of ultrasound power, and a 2 mm particle size. Under the optimal conditions, the corresponding response value predicted for SDF production was 39.52%, which was confirmed by validation experiments (n:5). The findings of this study demonstrate that the utilisation of UAE not only enhances yields but also enables the extraction of 10% higher soluble fibre content compared to conventional extraction methods, with reduced extraction times.