Termitomyces heimii mushroom is seasonal in which they only grow during the rainy season, it is necessary to establish a proper cultivation method so that the valuable bioactive compound can be produced at any time. Termitomyces heimii RFES 230622 (THR2), a wild-termite mushroom that was isolated from Negeri Sembilan during the monsoon, was sequenced and found to be 99% identical to the Termitomyces heimii strain, which was further supported by the mushroom's evolutionary distance (Knuc). The stipe, which seems to be cylindrical, creamy in colour, stuffed with thick annulus, and growing in the comb of termite nest, is the observable morphological trait of THR2. The isolated mushrooms were employed in tissue culture to grow mycelium on modified agar medium, and it was found that the PDA + ME + YE combination produced thick, full-plate mycelium growth after 14 days of incubation. Fruiting bodies, mycelial biomass, and cultured broth of THR2 were used to make extracts for antibacterial, antioxidant, antifungal analysis and determination of β-glucan polysaccharides. After 7 days of incubation, the THR2 mycelium was able to grow and generate polysaccharide in submerged liquid fermentation (SLF), with mycelial biomass of 8.55 g/L. Various extracts from fruiting bodies (ENS), mushroom mycelium (IPS), and culture broth were made (EPS). The THR2 fruiting bodies yield HW-ENS and CW-ENS, with corresponding polysaccharide contents of 3.20 g/L and 1.36 g/L, after hot-water and cold water extraction. Meanwhile, the hot-water extraction of mycelium produces IPS with 0.80 g/L and the extracellular polysaccharide (EPS) obtained was 1.44 g/L. The polysaccharides were verified as β-glucan by FTIR spectra analysis. These β-glucan exhibits antimicrobial properties with highest response observed in the β-glucan extracted from fruiting bodies (HW-ENS) compared to the β-glucan extracted from the mycelium whereas there was no zone of inhibition against Aspergillus niger which means it does not exhibit antifungal properties. All of the THR2 extract was found to contain phytochemicals like flavonoids, saponins, and terpenoids. Total flavonoid content (TFC), which was determined using quercetin as the standard, ranged from 2.16 to 2.86 mg/g in each THR2 extract. However, the highest concentration of flavonoid content was found in HW-ENS which is 2.86 ± 0.15. Using the DPPH assay, the antioxidant activity of THR2 extracts was evaluated, and the IC50 value was calculated. All extracts had IC50 values that range from 4.44 mg/ml to 6.71 mg/ml. HW-ENS sample of THR2 extract shows the least IC50 value which is 4.44 mg/ml whereas EPS showed the highest IC50 value which is 6.71 mg/ml. Out of this four THR2 extracts HW-ENS can be considered as extract with high potential of antioxidant activity as the IC50 value was the lowest with high percentage of inhibition at 10 mg/ml which was 90.83% compared to the other extracts.

Termitomyces sp. mushroom is an edible mushroom that belongs to the family of Lyophyllaceae and exhibited growth associated with termites in a symbiotic environment. In this study, Termitomyces sp. mushroom was isolated from several places located in Negeri Sembilan, Malaysia namely TM 1 (Senaling, Kuala Pilah, TM 2 (Batu Kikir, Kuala Pilah) and TM 3 (Rembau Negeri Sembilan). The collection process was conducted during the early rainy seasons within October to November in which these mushrooms were actively growing. The collected mushrooms fruiting bodies from the wild were further used in tissue culture for the growth of mycelium on the agar medium. Out of the three samples only TM 1 mycelium were successfully grown on the agar medium and subsequent optimization were carried out to enhance the growth of the TM 1 mycelium. The composition of the agar medium was manipulated, and it was observed that the combination of PDA (potato dextrose agar) + ME (malt extract) + YE (yeast extract) showed a thick full plate growth of mycelium in 14 days of cultivation. The mushroom mycelium of TM 1 was able to grow and produce polysaccharide in batch submerged liquid fermentation with a biomass of 8.55 g/L, intracellular polysaccharide (IPS CWE and IPS HWE) of 1.36 and 3.20 g/L respectively and extracellular polysaccharide of 1.44 g/L.

During the monsoon season, a wild-termite mushroom Termitomyces heimii RFES 230662 (THR2) was successfully isolated from Negeri Sembilan, Malaysia. The mushroom was morphologically labelled based on its stipe, pileus, and the budding mycelia from the termites nest. Genetic component of THR2 (300 bp) was sequenced and found to be 99% identical to Termitomyces sp. strain. The evolutionary distance (Knuc) of the isolate justified THR2 as T. heimii species. Two THR2 extracts were prepared from fruiting body (FB) and mycelial biomass (MB) for antimicrobial responses. THR2 was cultivated in a submerged-fermentation (SF) producing 8.55 g/L of MB, 0.80 g/L of endopolysaccharide (ENS) and 1.44 g/L of exopolysaccharide (EPS), respectively. For THR2-FB, two ENS extracts were obtained using hot (3.20 g/L) and cold-water (1.36 g/L) treatments. FTIR spectra analysis verified all polysaccharide as β-glucan when compared to laminarin standard. Those β-glucan possess antibacterial properties with highest response shown by ß-glucan-FB compared to ß-glucan-MB extracts. These findings serve as the blueprint for the production of β-glucan from a rare termite mushroom.