Polysaccharide with anticancer activity from Grifola frondosa cultured in industrial wastewater of Agaricus bisporus.

Culture conditions for Grifola frondosa using Agaricus bisporus industrial wastewater as a medium were optimized using Plackett-Burman and Box-Behnken methodologies. Plackett-Burman screening identified culture temperature, shaking speed, and wastewater solubility as the key factors influencing G. frondosa biomass. The Box-Behnken design then established optimal fermentation conditions: 23.7 °C, 202 rpm shaking speed, 10.4 % wastewater solubility, pH 6, 150 mL culture volume in a 250 mL flask, 9-day culture duration, and 10 % inoculation volume. Polysaccharides were isolated and purified from both the mycelia and the culture medium. UV and molecular weight analysis determined that the polysaccharides AIPGF01, NEPGF01, AEPGF01, and NIPGF01 were homogeneous, with molecular weights of 234.7 kDa, 125.2 kDa, 80.0 kDa, and 48.6 kDa, respectively. GC revealed that NIPGF01 and AIPGF01 contained only glucose, while NEPGF01 and AEPGF01 comprised both mannose and glucose in ratios of 12.37:83.69 and 10.06:85.78, respectively. FT-IR confirmed the presence of β-glycosidic linkages in all polysaccharides. NIPGF01 exhibited strong anticancer activity, significantly inhibiting human gastric cancer (MGC80-3) and liver cancer (Hep G2) cells. Apoptosis rates at 50 μg/mL, 200 μg/mL, and 800 μg/mL of NIPGF01 were 38.5 ± 3.5 %, 61.4 ± 4.3 %, and 79.2 ± 5.5 %, respectively. Flow cytometry indicated that NIPGF01 induced apoptosis in MGC80-3 cells by arresting them in the S and G₂ phases, leading to a marked reduction in G₁ phase cells.