Increasing soy isoflavonoid content and diversity by simultaneous malting and challenging by a fungus to modulate estrogenicity

Soybeans were germinated on a kilogram-scale, by the application of malting technology used in the brewing industry, and concomitantly challenged with Rhizopus microsporus var. oryzae. In a time-course experiment, samples were taken every 24 h for 10 days, and the isoflavonoid profile was analyzed by RP-UHPLC-MS. Upon induction with R. microsporus, the isoflavonoid composition changed drastically with the formation of phytoalexins belonging to the subclasses of the pterocarpans and coumestans and by prenylation of the various isoflavonoids. The pterocarpan content stabilized at 2.24 mg of daidzein equivalents (DE) per g after ～9 days. The levels of the less common glyceofuran, glyceollin IV, and V/VI ranged from 0.18 to 0.35 mg DE/g and were comparable to those of the more commonly reported glyceollins I, II, and III (0.22-0.32 mg DE/g) and glycinol (0.42 mg DE/g). The content of prenylated isoflavones after the induction process was 0.30 mg DE/g. The total isoflavonoid content increased by a factor of 10-12 on DW basis after 9 days, which was suggested to be ascribable to de novo synthesis. These changes were accompanied by a gradual increase in agonistic activity of the extracts toward both the estrogen receptor α (ERα) and ERβ during the 10-day induction, with a more pronounced activity toward ERβ. Thus, the induction process yielded a completely different spectrum of isoflavonoids, with a much higher bioactivity toward the estrogen receptors. This, together with the over 10-fold increase in potential bioactives, offers promising perspectives for producing more, novel, and higher potency nutraceuticals by malting under stressed conditions.