Metabolomics revealed novel isoflavones and optimal cultivation time of Cordyceps militaris fermentation.

Germinated soybean (GS) cultivated with Cordyceps militaris (GSC) might be a promising efficacious source of novel bioactive compounds. In this study, the metabolome changes between GS and GSC were investigated by liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) analysis coupled with a multivariate data set. Principal component analysis (PCA) and orthogonal projection to latent structures discriminate analysis (OPLS-DA) of GSC clearly showed higher levels of soyasaponin Bd, soyasaponin Bc(II), daidzein, genistein, four isoflavones (compounds 1-4), glycerol, proline, glutamine, pentitol, fructose, inositol, octadecanoic acid, and sucrose together with lower levels of pyroglutamic acid, citric acid, histidine, and palmitic acid in GSC than in GS. The structures of compounds 1-4 were analyzed by mass and NMR spectroscopy and were determined to be novel isoflavone methyl-glycosides (daidzein 7-O-beta-d-glucoside 4''-O-methylate (1), glycitein 7-O-beta-d-glucoside 4''-O-methylate (2), genistein 7-O-beta-d-glucoside 4''-O-methylate (3), and genistein 4'-O-beta-d-glucoside 4''-O-methylate (4)). Multivariate statistical models showed that metabolic changes of GSC were maximal within 1 week after the C. militaris inoculation, consistent with the strongest antioxidant activity of GSC cultivated for 1 week. This metabolomics study provides valuable information in regard to optimizing the cultivation process for bioactive compound production and describes an efficient way to screen for novel bioactive compounds from GSC.