Green mechanical and microbial processing of fungal mycelium for yogurt alternative production

• High-pressure homogenization enhanced viscoelasticity of fungal mycelium. • Particle size reduction and increased protein solubility improved gel structure. • Yogurt starter cultures enabled rapid acidification and improved rheology. • W. confusa VIII40 and P. claussenii 55 T produced high levels of EPS in situ . • EPS production enhanced water retention, viscosity, and gel network strength. This study presents a green, integrated strategy for valorizing fungal mycelium from Phellinus linteus ( PL ) and Cordyceps sinensis ( CS ) into functional yogurt alternatives. The effects of high-pressure homogenization (HPH), thermal pasteurization, and microbial fermentation on structural constituents and rheological properties were systematically investigated. HPH reduced particle size (by 77% in PL , 67% in CS ) and increased protein solubility (35.7% in PL , 42.7% in CS ), promoting the transition from a liquid-like system to a viscoelastic network. Prolonged pasteurization (85 °C, 30 min) enhanced starch gelatinization compared to shorter treatment (5 min), improving viscosity, water-binding capacity, and microbial safety. Fermentation with commercial yogurt starters reached pH 4.5 within 7 h and probiotic levels of 8–9 log colony-forming units (CFU)/g. In situ production of dextran and β-glucan by Weissella confusa VIII40 and Pediococcus claussenii 55 T, respectively, significantly enhanced viscosity, viscoelastic moduli, and creep-recovery behavior. Notably, high dextran yields (2.6–3.1%) significantly reduced syneresis to below 10%. Overall, the integrated mechanical and microbial processing demonstrates a clean-label method for converting underutilized fungal biomass into high fiber yogurt alternatives with improved rheological and textural quality.