Optimisation of Culture Conditions for Lignosus Rhinocerus Growth, Quantification of β-Glucan, Total Phenolic and Flavonoid Content, and α-Amylase Inhibitory Activities

Kong, Jessie Zhi Wei (2026) Optimisation of Culture Conditions for Lignosus Rhinocerus Growth, Quantification of β-Glucan, Total Phenolic and Flavonoid Content, and α-Amylase Inhibitory Activities. Final Year Project (Bachelor), Tunku Abdul Rahman University of Management and Technology.

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Abstract

Lignosus rhinocerus, also known as tiger milk mushroom, is a traditional medicinal fungus used by the Orang Asli to treat ailments such as coughing and asthma. It belongs to the Polyporaceae family, division Basidiomycota, under the genus Lignosus. Due to its slow growth in tropical forests, it is rare and valuable, prompting interest in laboratory cultivation to enhance bioactive compound production under controlled conditions. This study aimed to optimize culture conditions for L. rhinocerus and quantify its β-glucan, total phenolic content (TPC), total flavonoid content (TFC), and antidiabetic (α-amylase inhibitory) activity. Mycelium was first cultured on PDA agar for 10 days, then inoculated (15 plugs, 5 mm diameter) into 200 mL of PDB and Malt+ broth, and incubated at 120 rpm and 31.5 °C for 2 weeks. Cultures were harvested, freeze-dried, and ground into powder after liquid nitrogen treatment. For sclerotia production, 20 mL mycelium from Malt+ media was transferred into sawdust and brown rice substrates and incubated for 3 weeks in dark conditions (50–70% moisture), followed by incubation in soil for 3 months. DNA sequencing and BLAST analysis confirmed that mycelium from PDB media (87% query coverage, 93.8% identity), mycelium from Malt+ media (94% coverage, 98.62% identity), and sclerotia from sawdust substrate (88% coverage, 94.91% identity) were consistent with L. rhinocerus. However, sclerotia from brown rice substrate showed low similarity (26% coverage, 89.94% identity) and matched Aspergillus terreus, indicating contamination and exclusion from further assays. For the bioactivity assays, sclerotia from sawdust substrate had the highest TFC (105.93 ± 14.66 mgQE/g), TPC (73.76 ± 2.01 mgGAE/g), and α-amylase inhibitory activity (87.20 ± 0.88%), (P<0.05) indicating strong bioactivity. It also recorded higher (P<0.05) β-glucan content of 16.54 ± 2.21 mg/mL as compared to mycelium from PDB media which exhibited a significant lower content of β-glucan (6.87 ± 1.42 mg/mL). The β-glucan content in mycelium from Malt+ media (18.54 ± 4.26 mg/mL) is comparable (P>0.05) to the content from sclerotia from sawdust substrate. The mycelium yield obtained from culture using PDB media was 16.16 ± 1.32% while sclerotia from sawdust substrate recorded the lowest yield (2.56 ± 0.24%) due to slow development. Overall, Malt+ and sawdust substrates enhanced bioactive compound accumulation in L. rhinocerus, whereas PDB media leads to biomass production. The output of the project leads to bioactive-rich sclerotia production and aligned to United Nations Sustainable Development Goal 3 on Good Health and Well-Being.