The interaction between sucrose, yeast extract and initial pH was investigated to optimize critical medium components for mycelium biomass and production of exopolysaccharide (EPS) of Lentinus squarrosulus using Response Surface Methodology (RSM). A central composite design (CCD) was applied and a polynomial regression model with quadratic term was used to analyse the experimental data using analysis of variance (ANOVA). ANOVA analysis showed that the model was very significant (p < 0.0001) for both mycelium biomass and EPS production. The yeast extract concentrations and initial pH showed the strongest effect (p < 0.001) meanwhile sucrose concentrations showed significant value at p < 0.005 for mycelium biomass. For EPS production, only sucrose and yeast extract concentrations gave significant value with p < 0.001 and p < 0.005 respectively. The model was validated by applying the optimized conditions and 25.8 ± 0.9 g/L of mycelium biomass, and 5.7 ± 0.4 mg/mL EPS concentration was obtained. The estimated optimum conditions of the variables for the production of mycelium biomass and EPS production by Lentinus squarrosulus are as follows: sucrose concentration 114.61 g/L, yeast extract 1.62 g/L and initial pH of 5.81; sucrose concentration 115.8 g/L, yeast extract of 3.39 g/L and initial pH of 6.44 respectively.

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Various crude extracts of Lentinus squarrosulus mushroom mycelium and culture broth were prepared for antioxidant studies, glucan and carbohydrate compound analysis. Crude extract from ethanol, cold and hot water extraction was tested on several complementary test systems namely DPPH free radical scavenging, β-carotene linoleic acid oxidation, total phenolic compound, total flavonoid compound, total carbohydrate content and total glucan compositions. The antioxidant activity of the crude extracts of Lentinus squarrosulus mushroom mycelium and culture broth was determined by 2,2' diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method and β-carotene linoleic acid assay. Crude extracted from culture broth EPS EE recorded the highest radical scavenging effects at 20 mg/mL (85%) which is closer to the control antioxidants (ascorbic acid 97%; BHT 92.4%; BHA 93.6%). Both the ethanol extract (IPS EE and EPS EE) exhibited the highest β-carotene linoleic acid oxidation by recording IC50 values of 0.6 and 0.65 mg/mL respectively. IPS EE (0.59 mg/g) and EPS WE (0.58 mg/g) showed high flavonoid content whereas total phenol content was high in EPS WE (0.85 mg/g) and IPS EE (0.84 mg/g). Total carbohydrate concentrations were high in IPS HWE and IPS CWE with 19.1 and 16.3 mg/mL respectively and these data correlate with the β-glucan content in both extracts. The highest β-glucan content found in IPS CWE and IPS HWE were 12.2 and 11.4% w/w respectively.

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A statistical design approach has been used to optimize the production of biomass by Cunninghamella sp. 2A1, evaluated based on lipidless biomass. A 2 3 full factorial central composite design (CCD) was chosen to study the combined effects of three factors; ammonium tartrate, peptone and glucose concentrations. The p-value for each factors was < 0.05 suggesting that these factors have significant effect on the production of lipidless biomass. The production is represented by a linear model with p-value < 0.0001. The optimized medium consists of 3.86 g/l ammonium tartrate, 55.84 g/l glucose and 7.73 g/l peptone predicted 16.83 g/l lipidless biomass. Results from four replications based on the optimized medium produced 18.48 g/l lipidless biomass, which are in close agreement with the predicted value. The coefficient for glucose was the highest indicating it as the most significant factor affecting lipidless biomass production.

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