{Lentinula edodes (shiitake) is an edible mushroom that contains many constituents and β‐glucan is considered a major polysaccharide. This study, therefore, aimed to characterize β‐glucan and evaluate its activities. Fresh fruit bodies of L. edodes were used for β‐glucan extraction and followed by analyses via Fourier transform infrared, nuclear magnetic resonance and high performance liquid chromatography confirming its characteristic structure. For evaluating biological activities of β‐glucan, different concentrations (0·1–3·5 mg ml−1) were assessed. The optimal dose was 3·5 mg ml−1 that showed the highest scavenging radical ability (75·3\%) confirms antioxidant activity, strong inhibition of the peroxyl radical (80·9\%) to inhibit lipid peroxidation, elevation the inhibition percentage of both α‐amylase (73·4\%) and α‐glucosidase (70·3\%) indicates the antidiabetic properties, and highest AFB1 reduction (88\%) which ensured the aflatoxin‐detoxifying ability. In addition, antifungal activity of β‐glucan was evaluated to inhibit sporulation process in Aspergillus niger and recorded with minimum inhibitory concentration of 2·5 mg ml−1 and minimum fungicidal concentration of 3 mg ml−1. In a dose‐dependent manner, higher concentration of β‐glucan affects viability of tumour cells concomitant induces potent anti‐cancer immune responses and inhibited the activity of topoisomerase I which are considered an important target for cancer chemotherapy. Therefore, L. edodes‐β‐glucan has the potential to act as a suggestive agent for antioxidant, antidiabetic, antifungal activity and aflatoxin detoxification.Significance and Impact of the Study: The isolated β‐glucan was demonstrated as a potential molecule that could be used as an anti‐oxidant, anti‐lipid peroxidation, anti‐diabetic, anti‐fungal, aflatoxin‐detoxifying and anti‐tumour agent for biomedical applications. }
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