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Lentinula edodes is an edible mushroom known for its medicinal properties attributed to different secondary metabolites. In this article, the effect of glucose concentration, aeration and agitation on the production of biomass and intrametabolites in submerged fermentation was evaluated. By means of a Box-Behnken design, these variables were optimized to obtain a multifunctional biomass. It was determined that both glucose concentration and agitation had a significant effect on the response variables (biomass, sterols and polysaccharides). Under the evaluated conditions, the values of the variables that optimize biomass production were 1.2 vvm, 60 rpm and 21.97 g / L of glucose. Subsequently, the sterols and total carbohydrates extracted from the biomass were quantified through a spectrophotometric analysis. To optimize these two response variables, similar values are required for aeration (1.2 vvm) and agitation (60 rpm), while the glucose concentration was 16.32 g / L and 19.6 g / L for sterols and total polysaccharides respectively.