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Wild-cultivated medicinal mushroom Ganoderma lucidum was morphologically identified and sequenced using phylogenetic software. In submerged-liquid fermentation (SLF), biomass, exopolysaccharide (EPS) and intracellular polysaccharide (IPS) production of the identified G. lucidum was optimised based on initial pH, starting glucose concentration and agitation rate parameters using response surface methodology (RSM). Molecularly, the G. lucidum strain QRS 5120 generated 637 base pairs, which was commensurate with related Ganoderma species. In RSM, by applying central composite design (CCD), a polynomial model was fitted to the experimental data and was found to be significant in all parameters investigated. The strongest effect ( p < 0.0001) was observed for initial pH for biomass, EPS and IPS production, while agitation showed a significant value ( p < 0.005) for biomass. By applying the optimized conditions, the model was validated and generated 5.12 g/L of biomass (initial pH 4.01, 32.09 g/L of glucose and 102 rpm), 2.49 g/L EPS (initial pH 4, 24.25 g/L of glucose and 110 rpm) and 1.52 g/L of IPS (and initial pH 4, 40.43 g/L of glucose, 103 rpm) in 500 mL shake flask fermentation. The optimized parameters can be upscaled for efficient biomass, EPS and IPS production using G. lucidum .

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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.

The pellet morphology and diameter range (DR) of Ganoderma lucidum were observed in a repeated-batch fermentation (RBF) for the trio total production of biomass, exopolysaccharide (EPS) and endopolysaccharide (ENS). Two factors were involved in RBF; broth replacement ratio (BRR: 60%, 75% and 90%) and broth replacement time point (BRTP: log, transition and stationary phase) in days. In RBF, 34.31 g/L of biomass favoured small-compact pellets with DR of 20.67 µm< d < 24.00 µm (75% BRR, day 11 of BRTP). EPS production of 4.34 g/L was prone to ovoid-starburst pellets with DR of 34.33 µm< d <35.67 µm (75% BRR, day 13 of BRTP). Meanwhile, the highest 2.43 g/L of ENS production favoured large-hollow pellets with DR of 34.00 µm< d < 38.67 µm (90% BRR, day 13 of BRTP). In addition, RBF successfully shortened the biomass-EPS–ENS fermentation period (31, 33 and 35 days) from batch to 5 days, in seven consecutive cycles of RBF. In a FTIR detection, β-glucan (BG) from EPS and ENS extracts were associated with β-glycosidic linkages (2925 cm −1 , 1635 cm −1 , 1077 cm −1 , 920 cm −1 and 800 cm −1 wavelengths) with similar 1 H NMR spectral behaviour (4.58, 3.87 and 3.81 ppm). Meanwhile, 4 mg/L of BG gave negative cytotoxic effects on normal gingival cell line (hGF) but induced antiproliferation (IC 50 = 0.23 mg/mL) against cancerous oral Asian cellosaurus cell line (ORL-48). Together, this study proved that G. lucidum mycelial pellets could withstand seven cycles of long fermentation condition and possessed anti-oral cancer beta-glucan, which suits large-scale natural drug fermentation.

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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