Evaluation of Three-Phase Partitioning for Efficient and Simultaneous Isolation of Immunomodulatory Polysaccharides and Proteins from Lentinula Edodes Mushroom

Lentinula edodes is one of the most popular edible mushrooms and is also widely used as a health food. Polysaccharides (PS) and proteins are two major groups of biomacromolecule constituents contributing to the health benefits of L. edodes. However, previous studies have mainly devoted to the PS and paid much less attention to the proteins. This study was to evaluate the efficacy of three-phase partitioning (TPP) method for isolating PS and protein components from L. edodes mushrooms and to assess their molecular properties and biological activities. The TPP conditions were optimized by response surface methodology (RSM) as 48.0% (w/v) ammonium sulfate (NH₄)₂SO₄, 1.2 volume ratio of t-butanol to the crude extract solution, and 52 °C temperature, giving a maximum PS yield of 11.64% and a protein yield of 2.06%. The PS was composed mainly of glucose, galactose, and mannose with a molecular weight of 7.35 kDa. Both the PS- and protein-rich fractions exhibited notable immunostimulatory activity, as evidenced by their ability to enhance the NO production and the phagocytic capacity of RAW 264.7 cells. Notably, the protein-rich fraction demonstrated particularly remarkable activity with a low EC50 value of 0.28 μg/mL. The results suggested that TPP is a simple and effective method for simultaneous isolation of bioactive PS and proteins from mushrooms.